Chemical compounds

ABSTRACT

The invention concerns compounds of Formula (I) 
                         
or pharmaceutically-acceptable salts thereof, wherein R 1  to R 5  have any of the meanings defined hereinbefore in the description; processes for their preparation, pharmaceutical compositions containing them and their use in the treatment of cell proliferative disorders.

This application is a continuation of U.S. patent application Ser. No.14/840,342, filed Aug. 31, 2015 which is a continuation of U.S. patentapplication Ser. No. 14/287,332, filed May 27, 2014, granted Oct. 13,2015 as U.S. Pat. No. 9,155,727, which claims the benefit under 35U.S.C. § 119(e) of Provisional Patent Application No. 61/827,951, filedon May 28, 2013, and Provisional Patent Application No. 61/915,685,filed on Dec. 13, 2013.

The invention concerns certain novel indole derivatives, orpharmaceutically-acceptable salts thereof, which possess anti-canceractivity and are accordingly potentially useful in methods of treatmentof the human or animal body. The invention also concerns processes forthe manufacture of said indole derivatives, pharmaceutical compositionscontaining them and their use in therapeutic methods, for example in themanufacture of medicaments for use in the prevention or treatment ofcancers in a warm-blooded animal such as man, including use in theprevention or treatment of cancer.

The present invention also relates to indole derivatives that areselective down-regulators of the estrogen receptor.

Estrogen receptor alpha (ERα, ESR1, NR3A) and estrogen receptor beta(ERβ, ESR2, NR3b) are steroid hormone receptors which are members oflarge nuclear receptor family. Structured similarly to all nuclearreceptors, ERa is composed of six functional domains (named A-F)(Dahlman-Wright, et al., Pharmacol. Rev., 2006, 58:773-781) and isclassified as a ligand-dependent transcription factor because after itsassociation with the specific ligand, the female sex steroid hormone 17bestradiol (E2), the complex binds to genomic sequences, named EstrogenReceptor Elements (ERE) and interacts with co-regulators to modulate thetranscription of target genes. The ERα gene is located on 6q25.1 andencodes a 595AA protein and multiple isoforms can be produced due toalternative splicing and translational start sites. In addition to theDNA binding domain (Domain C) and the ligand binding domain (Domain E)the receptor contains an N-terminal (A/B) domain, a hinge (D) domainthat links the C and E domains and a C-terminal extension (F domain).While the C and E domains of ERa and ERb are quite conserved (96% and55% amino acid identity respectively) conservation of the A/B, D and Fdomains is poor (below 30% amino acid identity). Both receptors areinvolved in the regulation and development of the female reproductivetract and in addition play roles in the central nervous system,cardiovascular system and in bone metabolism. The genomic action of ERsoccurs in the nucleus of the cell when the receptor binds EREs directly(direct activation or classical pathway) or indirectly (indirectactivation or non-classical pathway). In the absence of ligand, ERs areassociated with heat shock proteins, Hsp90 and Hsp70, and the associatedchaperone machinery stabilizes the ligand binding domain (LBD) making itaccessible to ligand. Liganded ER dissociates from the heat shockproteins leading to a conformational change in the receptor that allowsdimerisation, DNA binding, interaction with co-activators orco-repressors and modulation of target gene expression. In thenon-classical pathway, AP-1 and Sp-1 are alternative regulatory DNAsequences used by both isoforms of the receptor to modulate geneexpression. In this example, ER does not interact directly with DNA butthrough associations with other DNA bound transcription factors e.g.c-Jun or c-Fos (Kushner et al., Pure Applied Chemistry 2003,75:1757-1769). The precise mechanism whereby ER affects genetranscription is poorly understood but appears to be mediated bynumerous nuclear factors that are recruited by the DNA bound receptor.The recruitment of co-regulators is primarily mediated by two proteinsurfaces, AF2 and AF1 which are located in E-domain and the A/B domainrespectively. AF1 is regulated by growth factors and its activitydepends on the cellular and promoter environment whereas AF2 is entirelydependent on ligand binding for activity. Although the two domains canact independently, maximal ER transcriptional activity is achievedthrough synergistic interactions via the two domains (Tzukerman, et al.,Mol. Endocrinology, 1994, 8:21-30). Although ERs are consideredtranscription factors they can also act through non-genomic mechanismsas evidenced by rapid ER effects in tissues following E2 administrationin a timescale that is considered too fast for a genomic action. It isstill unclear if receptors responsible for the rapid actions of estrogenare the same nuclear ERs or distinct G-protein coupled steroid receptors(Warner, et al., Steroids 2006 71:91-95) but an increasing number of E2induced pathways have been identified e.g. MAPK/ERK pathway andactivation of endothelial nitric oxide synthase and PI3K/Akt pathway. Inaddition to ligand dependent pathways, ERα has been shown to have ligandindependent activity through AF-1 which has been associated withstimulation of MAPK through growth factor signalling e.g. insulin likegrowth factor 1 (IGF-1) and epidermal growth factor (EGF). Activity ofAF-1 is dependent on phosphorylation of Ser118 and an example ofcross-talk between ER and growth factor signalling is thephosphorylation of Ser 118 by MAPK in response to growth factors such asIGF-1 and EGF (Kato, et al., Science, 1995, 270:1491-1494).

A large number of structurally distinct compounds have been shown tobind to ER. Some compounds such as endogenous ligand E2, act as receptoragonists whereas others competitively inhibit E2 binding and act asreceptor antagonists. These compounds can be divided into 2 classesdepending on their functional effects. Selective estrogens receptormodulators (SERMs) such as tamoxifen have the ability to act as bothreceptor agonists and antagonists depending on the cellular and promotercontext as well as the ER isoform targeted. For example tamoxifen actsas an antagonist in breast but acts as a partial agonist in bone, thecardiovascular system and uterus. All SERMs appear to act as AF2antagonists and derive their partial agonist characteristics throughAF1. A second group, fulvestrant being an example, are classified asfull antagonists and are capable of blocking estrogen activity via thecomplete inhibition of AF1 and AF2 domains through induction of a uniqueconformation change in the ligand binding domain (LBD) on compoundbinding which results in complete abrogation of the interaction betweenhelix 12 and the remainder of the LBD, blocking co-factor recruitment(Wakeling, et al., Cancer Res., 1991, 51:3867-3873; Pike, et al.,Structure, 2001, 9:145-153).

Intracellular levels of ERα are down-regulated in the presence of E2through the ubiquitin/proteosome (Ub/26S) pathway. Polyubiquitinylationof liganded ERα is catalysed by at least three enzymes; theubiquitin-activating enzyme E1 activated ubiquitin is conjugated by E2with lysine residues through an isopeptide bond by E3 ubiquitin ligaseand polyubiquitinated ERα is then directed to the proteosome fordegradation. Although ER-dependent transcription regulation andproteosome-mediated degradation of ER are linked (Lonard, et al., Mol.Cell, 2000 5:939-948), transcription in itself is not required for ERαdegradation and assembly of the transcription initiation complex issufficient to target ERα for nuclear proteosomal degradation. This E2induced degradation process is believed to necessary for its ability torapidly activate transcription in response to requirements for cellproliferation, differentiation and metabolism (Stenoien, et al., Mol.Cell Biol., 2001, 21:4404-4412). Fulvestrant is also classified as aselective estrogen receptor down-regulator (SERD), a subset ofantagonists that can also induce rapid down-regulation of ERα via the26S proteosomal pathway. In contrast a SERM such as tamoxifen canincrease ERα levels although the effect on transcription is similar tothat seen for a SERD.

Approximately 70% of breast cancers express ER and/or progesteronereceptors implying the hormone dependence of these tumour cells forgrowth. Other cancers such as ovarian and endometrial are also thoughtto be dependent on ERα signalling for growth. Therapies for suchpatients can inhibit ER signalling either by antagonising ligand bindingto ER e.g. tamoxifen which is used to treat early and advanced ERpositive breast cancer in both pre and post menopausal setting;antagonising and down-regulating ERα e.g. fulvestrant which is used totreat breast cancer in women which have progressed despite therapy withtamoxifen or aromatase inhibitors; or blocking estrogen synthesis e.g.aromatase inhibitors which are used to treat early and advanced ERpositive breast cancer. Although these therapies have had an enormouslypositive impact on breast cancer treatment, a considerable number ofpatients whose tumours express ER display de novo resistance to existingER therapies or develop resistance to these therapies over time. Severaldistinct mechanism have been described to explain resistance tofirst-time tamoxifen therapy which mainly involve the switch fromtamoxifen acting as an antagonist to an agonist, either through thelower affinity of certain co-factors binding to the tamoxifen-ERαcomplex being off-set by over-expression of these co-factors, or throughthe formation of secondary sites that facilitate the interaction of thetamoxifen-ERα complex with co-factors that normally do not bind to thecomplex. Resistance could therefore arise as a result of the outgrowthof cells expressing specific co-factors that drive the tamoxifen-ERαactivity. There is also the possibility that other growth factorsignalling pathways directly activate the ER receptor or co-activatorsto drive cell proliferation independently of ligand signalling.

More recently, mutations in ESR1 have been identified as a possibleresistance mechanism in metastatic ER-positive patient derived tumoursamples and patient-derived xenograft models (PDX) at frequenciesvarying from 17-25%. These mutations are predominantly, but notexclusively, in the ligand-binding domain leading to mutated functionalproteins; examples of the amino acid changes include Ser463Pro,Val543Glu, Leu536Arg, Tyr537Ser, Tyr537Asn and Asp538Gly, with changesat amino acid 537 and 538 constituting the majority of the changescurrently described. These mutations have been undetected previously inthe genomes from primary breast samples characterised in the CancerGenome Atlas database. Of 390 primary breast cancer samples positive forER expression not a single mutation was detected in ESR1 (Cancer GenomeAtlas Network, 2012 Nature 490: 61-70). The ligand binding domainmutations are thought to have developed as a resistance response toaromatase inhibitor endocrine therapies as these mutant receptors showbasal transcriptional activity in the absence of estradiol. The crystalstructure of ER, mutated at amino acids 537 and 538, showed that bothmutants favoured the agonist conformation of ER by shifting the positionof helix 12 to allow co-activator recruitment and thereby mimickingagonist activated wild type ER. Published data has shown that endocrinetherapies such as Tamoxifen and Fulvestrant can still bind to ER mutantand inhibit transcriptional activation to some extent and thatFulvestrant is capable of degrading Try537Ser but that higher doses maybe needed for full receptor inhibition (Toy et al., Nat. Genetics 2013,45: 1439-1445; Robinson et al., Nat. Genetics 2013, 45: 144601451; Li,S. et al. Cell Rep. 4, 1116-1130 (2013). It is therefore feasible thatcertain compounds of the formula (I), or pharmaceutically-acceptablesalts thereof will be capable of down-regulating and antagonising mutantER although it is not known at this stage whether ESR1 mutations areassociated with an altered clinical outcome.

Regardless of which resistance mechanism or combination of mechanismstakes place, many are still reliant on ER-dependent activities and thatremoval of the receptor through a SERD mechanism offers the best way ofremoving the ERα receptor from the cell. Fulvestrant is currently theonly SERD approved for clinical use, yet despite its mechanisticproperties, the pharmacological properties of the drug have limited itsefficacy due to the current limitation of a 500 mg monthly dose whichresults in less than 50% turnover of the receptor in patient samplescompared to the complete down-regulation of the receptor seen inin-vitro breast cell line experiments (Wardell, et al., Biochem. Pharm.,2011, 82:122-130). Hence there is a need for new ER targeting agentsthat have the required pharmaceutical properties and SERD mechanism toprovide enhanced benefit in the early, metastatic and acquiredresistance setting.

The compounds of the invention have been found to possess potentanti-tumour activity, being useful in inhibiting the uncontrolledcellular proliferation which arises from malignant disease. Thecompounds of the invention provide an anti-tumour effect by, as aminimum, acting as SERDs.

According to one aspect of the invention there is provided a compound ofthe Formula (I) or a pharmaceutically-acceptable salt thereof

wherein:

-   R¹ and R² are each independently H or F;-   R³ is H or methyl; and-   either:-   a) R⁴ is H and R⁵ is F; or-   b) R⁴ is F and R⁵ is H.

In another aspect of the invention, there is provided a compound ofFormula (I) as defined above.

The compounds of formula (I) have one, two or three chiral centres andthe invention encompasses pure chiral forms or mixtures thereof in anyproportion. The synthesis of optically active forms may be carried outby standard techniques of organic chemistry well known in the art, forexample by synthesis from optically active starting materials or byresolution of a racemic form. Similarly, the above-mentioned activitymay be evaluated using the standard laboratory techniques.

A particular enantiomer or diasteroeisomer of a compound describedherein may be more active than other enantiomers or diastereoisomers ofthe same compound.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, which is a single enantiomer being in an enantiomeric excess (%ee) of ≥95, ≥98% or ≥99%. Conveniently, the single enantiomer is presentin an enantiomeric excess (% ee) of ≥99%.

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a compound of the Formula(I), which is a single enantiomer being in an enantiomeric excess (% ee)of ≥95, ≥98% or ≥99% or a pharmaceutically-acceptable salt thereof, inassociation with a pharmaceutically-acceptable diluent or carrier.Conveniently, the single enantiomer is present in an enantiomeric excess(% ee) of ≥99%.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, which is a single diastereoisomer being in an diastereomericexcess (% de) of ≥95, ≥98% or ≥99%. Conveniently, the singlediastereoisomer is present in an diastereomeric excess (% de) of ≥99%.

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a compound of the Formula(I), which is a single diastereoisomer being in an diastereomeric excess(% de) of ≥95, ≥98% or ≥99% or a pharmaceutically-acceptable saltthereof, in association with a pharmaceutically-acceptable diluent orcarrier. Conveniently, the single diastereoisomer is present in andiastereomeric excess (% de) of ≥99%.

In one particular aspect, the compound of Formula (I) is a compound ofFormula (IA):

In another aspect, the compound of Formula (I) is a compound of Formula(IB):

Reference herein to compounds of Formula (I) is to be understood asreferring to compounds of Formula (IA) and/or (IB) unless statedotherwise.

For Example, the compound of Example 1,(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid, is an example of a compound of Formula (IA).

Its isomer,(E)-3-(3,5-difluoro-4-((1S,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid, is an example of a compound of Formula (IB).

Both of these two isomers are examples of(E)-3-(3,5-difluoro-4-((3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid, which is an example of a compound of Formula (I).

Some compounds of Formula (I) may be crystalline and may have more thanone crystalline form. It is to be understood that the present inventionencompasses any crystalline or amorphous form, or mixtures thereof,which form possesses properties useful in SERD activity, it being wellknown in the art how to determine efficacy of a crystalline or amorphousform for the SERD activity by the standard tests described hereinafter.

It is generally known that crystalline materials may be analysed usingconventional techniques such as X-Ray Powder Diffraction (hereinafterXRPD) analysis, Differential Scanning Calorimetry (hereinafter DSC),Thermal Gravimetric Analysis (hereinafter TGA), Diffuse ReflectanceInfrared Fourier Transform (DRIFT) spectroscopy, Near Infrared (NIR)spectroscopy, solution and/or solid state nuclear magnetic resonancespectroscopy. The water content of such crystalline materials may bedetermined by Karl Fischer analysis.

As an example, the compound of Example 7 exhibits crystallinity and onecrystalline form has been identified.

Accordingly, a further aspect of the invention is Form A of(E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (Example 7).

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at least one specific peak at about2-theta=4.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at least one specific peak at about2-theta=10.8°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at least two specific peaks at about2-theta=4.5 and 10.8°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at specific peaks at about 2-theta=4.5,4.8, 6.1, 7.9, 9.9, 10.8, 13.4, 14.0, 14.3 and 18.5°.

According to the present invention there is provided crystalline form,Form A of Example 7 which has an X-ray powder diffraction patternsubstantially the same as the X-ray powder diffraction pattern shown inFIG. 1.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at least one specific peak at about2-theta=4.5° plus or minus 0.2° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at least one specific peak at about2-theta=10.8° plus or minus 0.2° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at least two specific peaks at about2-theta=4.5 and 10.8° plus or minus 0.2° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of Example 7 which has an X-raypowder diffraction pattern with at specific peaks at about 2-theta=4.5,4.8, 6.1, 7.9, 9.9, 10.8, 13.4, 14.0, 14.3 and 18.5° plus or minus 0.2°2-theta.

Furthermore, Example 1 also shows crystallinity.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with at least onespecific peak at about 2-theta=8.4°.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with at least onespecific peak at about 2-theta=10.9°.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with at least twospecific peaks at about 2-theta=8.4° and 10.9°.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with specific peaksat about 2-theta=8.4, 10.9, 18.3, 24.0 and 14.0°.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with specific peaksat about 2-theta=8.4, 10.9, 18.3, 24.0, 14.0, 19.0, 14.4, 13.0, 15.3,20.6°.

According to the present invention there is provided crystalline form,Form B of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern substantially thesame as the X-ray powder diffraction pattern shown in FIG. 2.

According to the present invention there is provided crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with at least onespecific peak at 2-theta=8.4° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with at least onespecific peak at 2-theta=10.9° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with at least twospecific peaks at 2-theta=8.40 and 10.90 wherein said values may be plusor minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with specific peaksat about 2-theta=8.4, 10.9, 18.3, 24.0 and 14.0° wherein said values maybe plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid which has an X-ray powder diffraction pattern with specific peaksat 2-theta=8.4, 10.9, 18.3, 24.0, 14.0, 19.0, 14.4, 13.0, 15.3, 20.6°wherein said values may be plus or minus 0.2° 2-theta.

When it is stated that the present invention relates to a crystallineform of Example 1 Form B, the degree of crystallinity is convenientlygreater than about 60%, more conveniently greater than about 80%,preferably greater than about 90% and more preferably greater than about95%. Most preferably the degree of crystallinity is greater than about98%.

Furthermore, when it is stated that the present invention relates to acrystalline form of Example 1 Form B, the material is preferablysubstantially free of other crystalline forms or amorphous material. By“substantially free”, we mean conveniently greater than about 60%, moreconveniently greater than about 80%, preferably greater than about 90%,more preferably greater than about 95% and most preferably greater thanabout 98% of single polymorph.

It will be understood that 2-theta values of the X-ray powderdiffraction patterns may vary slightly from one machine to another orfrom one sample to another, and so the values quoted are not to beconstrued as absolute.

It is known that an X-ray powder diffraction pattern may be obtainedwhich has one or more measurement errors depending on measurementconditions (such as equipment or machine used). In particular, it isgenerally known that intensities in an X-ray powder diffraction patternmay fluctuate depending on measurement conditions. Therefore it shouldbe understood that the crystalline Forms of the present inventiondescribed above, unless otherwise stated, are not limited to thecrystals that provide X-ray powder diffraction patterns identical to theX-ray powder diffraction pattern shown in the relevant Figures, and anycrystals providing X-ray powder diffraction patterns substantially thesame as those shown in these Figures fall within the scope of thepresent invention. A person skilled in the art of X-ray powderdiffraction is able to judge the substantial identity of X-ray powderdiffraction patterns.

Persons skilled in the art of X-ray powder diffraction will also realisethat the relative intensity of peaks can be affected by, for example,grains above 30 microns in size and non-unitary aspect ratios, which mayaffect analysis of samples. The skilled person will also realise thatthe position of reflections can be affected by the precise height atwhich the sample sits in the diffractometer and the zero calibration ofthe diffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values (see Jenkins, R & Snyder, R. L. ‘Introductionto X-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C. W.(1948), Chemical Crystallography, Clarendon Press, London; Klug, H. P. &Alexander, L. E. (1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is approximately plus or minus 0.2° 2-theta, and suchdegree of a measurement error should be taken into account whenconsidering the X-ray powder diffraction data. Furthermore, it should beunderstood that intensities might fluctuate depending on experimentalconditions and sample preparation (preferred orientation).

Particular compounds of the invention are each of the Examples, each ofwhich provides a further independent aspect of the invention. Furtherparticular compounds of the invention are pharmaceutically-acceptablesalt(s) of each of the Examples, each of which provides a furtherindependent aspect of the invention.

According to a further aspect of the invention there is provided acompound of the Formula (I), which is obtainable by following any of theExamples as disclosed herein.

A further feature is any of the scopes defined herein with the provisothat specific Examples, such as Example 1, 2, 3 etc. are individuallydisclaimed.

It will be appreciated by those skilled in the art that certaincompounds of Formula (I) contain asymmetrically substituted carbonatoms, and accordingly may exist in, and be isolated in,optically-active and racemic forms. Some compounds of Formula (I) mayexhibit polymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic or stereoisomericform, or mixtures thereof, which form possesses properties useful asSERDs, it being well known in the art how to prepare optically-activeforms (for example, by resolution of the racemic form byrecrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, by enzymatic resolution, bybiotransformation, or by chromatographic separation using a chiralstationary phase) and how to determine efficacy as SERDs by the standardtests described hereinafter.

It is to be understood that certain compounds of Formula (I) definedabove may exhibit the phenomenon of tautomerism. It is to be understoodthat the present invention includes in its definition any suchtautomeric form, or a mixture thereof, which possesses SERD activity andis not to be limited merely to any one tautomeric form utilised withinthe formulae drawings or named in the Examples. In general, just one ofany such tautomeric forms is named in the Examples that followhereinafter or is presented in any relevant formulae drawings thatfollow hereinafter.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes will be understood toinclude those atoms having the same atomic number but different massnumbers. For example, isotopes of hydrogen include tritium anddeuterium. Isotopes of carbon include ¹³C and ¹⁴C. A deuterated versionof Example 1 is described in Example 10.

A suitable pharmaceutically-acceptable salt of a compound of the Formula(I) is, for example, an alkali or alkaline earth metal salt such as asodium, calcium or magnesium salt, or an ammonium salt, or a salt withan organic base such as methylamine, dimethylamine, trimethylamine,piperidine, morpholine or tris-(2-hydroxyethyl)amine. Further suitablepharmaceutically-acceptable salts of a compound of the Formula (I) maybe other metal salts, such as potassium, zinc, or other such metalcations known in the art. In one aspect of the invention, apharmaceutically-acceptable salt of a compound of Formula (I) is a saltwith a metal cation, an ammonium salt or a salt with an organic base.

A further suitable pharmaceutically-acceptable salt of a compound of theFormula (I) is, for example, a salt formed within the human or animalbody after administration of a compound of the Formula (I).

A suitable pharmaceutically-acceptable salt of a compound of the Formula(I) may also be, for example, an acid-addition salt of a compound of theFormula (I), for example an acid-addition salt with a strong inorganicor organic acid such as hydrochloric, hydrobromic, sulphuric ortrifluoroacetic acid. Other potential suitablepharmaceutically-acceptable salt of a compound of the Formula (I) mayalso be as described below for Example 1. In another aspect of theinvention, a pharmaceutically-acceptable salt of a compound of Formula(I) is an acid-addition salt.

Experiments looking at formation of salts of the compounds of Formula(I) examined the potential for Example 1((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid) to form crystalline salts. The following acids and bases weretried: acetic acid, adipic acid, benzene sulfonic acid, benzoic acid,cinnamic acid, citric acid, D,L-lactic acid, ethane disulfonic acid,ethane sulfonic acid, fumaric acid, hydrochloric acid, L-tartaric acid,maleic acid, malic acid, malonic acid, methane sulfonic acid,napadisylic acid, phosphoric acid, saccharin, succinic acid, sulphuricacid, toluene sulfonic acid, calcium acetate, diethylamine,ethanolamine, ethylenediamine, hydroxyethylpyrrolidine, magnesiumacetate, meglumine, piperazine, potassium hydroxide, sodium hydroxide,t-butylamine, triethanolamine, tris(hydroxymethyl)aminomethane (Tris)and N,N-diethylethanolamine.

Of the above acids and bases, isolatable solid salts were not alwaysobtainable, or not obtainable in crystalline form in the experimentalconditions employed. Preferred salts of Example 1 include those whichmay be isolated in crystalline form, for example, benzene sulfonic acidsalt (besylate salt), succinic acid salt (succinate salt) and maleicacid salt (maleate salt).

In one aspect, suitable salts of Example 1 may include besylate,succinate and maleate. In another aspect, a suitable salt of Example 1may be the maleate salt, which is described in Example 11.

It is further to be understood that a suitablepharmaceutically-acceptable co-crystal of a compound of the Formula (I)also forms an aspect of the present invention. For the avoidance ofdoubt, the term co-crystal (or cocrystal) refers to a multicomponentsystem in which there exists a host API (active pharmaceuticalingredient) molecule or molecules and a guest (or co-former) molecule ormolecules. In a co-crystal, both the API molecule and the guest (orco-former) molecule exist as a solid at room temperature when alone intheir pure form (in order to distinguish the co-crystal from solvates orhydrates). Salts, in which significant or complete proton exchangeoccurs between the API molecule and the guest molecule, are excludedfrom this particular definition. In a co-crystal, the API and co-formermolecules interact by hydrogen bonding and possibly other non-covalentinteractions. Pharmaceutically acceptable co-formers include neutralmolecules such as nicotinamide, resorcinol and xylenols, as well asionisable molecules such as oxalic acid, 3,5-dihydroxybenzoic acid andisoquinoline (the extent of proton exchange determining whether a saltor co-crystal is formed). It may be noted that a co-crystal may itselfform solvates, including hydrates.

It is further to be understood that a suitablepharmaceutically-acceptable solvate of a compound of the Formula (I)also forms an aspect of the present invention. A suitablepharmaceutically-acceptable solvate is, for example, a hydrate such as ahemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate or analternative quantity thereof.

It is further to be understood that a suitablepharmaceutically-acceptable pro-drug of a compound of the Formula (I)also forms an aspect of the present invention. Accordingly, thecompounds of the invention may be administered in the form of apro-drug, which is a compound that is broken down in the human or animalbody to release a compound of the invention. A pro-drug may be used toalter the physical properties and/or the pharmacokinetic properties of acompound of the invention. A pro-drug can be formed when the compound ofthe invention contains a suitable group or substituent to which aproperty-modifying group can be attached. Examples of pro-drugs includein-vivo cleavable ester derivatives that may be formed at a carboxygroup in a compound of the Formula (I).

Accordingly, the present invention includes those compounds of theFormula (I) as defined hereinbefore when made available by organicsynthesis and when made available within the human or animal body by wayof cleavage of a pro-drug thereof. Accordingly, the present inventionincludes those compounds of the Formula (I) that are produced by organicsynthetic means and also such compounds that are produced in the humanor animal body by way of metabolism of a precursor compound, that is acompound of the Formula (I) may be a synthetically-produced compound ora metabolically-produced compound.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula (I) is one that is based on reasonable medical judgement asbeing suitable for administration to the human or animal body withoutundesirable pharmacological activities and without undue toxicity.

Various forms of pro-drug have been described, for example in thefollowing documents:—

-   a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder,    et al. (Academic Press, 1985);-   b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);-   c) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and    Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991);-   d) H. Bundgaard, Advanced Drug Delivery Reviews. 8, 1-38 (1992);-   e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285    (1988);-   f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984);-   g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”,    A.C.S. Symposium Series, Volume 14; and-   h) E. Roche (editor), “Bioreversible Carriers in Drug Design”,    Pergamon Press, 1987.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula I that possesses a carboxy group is, for example, an in vivocleavable ester thereof. An in vivo cleavable ester of a compound of theFormula I containing a carboxy group is, for example, apharmaceutically-acceptable ester which is cleaved in the human oranimal body to produce the parent acid. Suitablepharmaceutically-acceptable esters for carboxy include (1-6C)alkylesters such as methyl, ethyl and tert-butyl, (1-6C)alkoxymethyl esterssuch as methoxymethyl esters, (1-6C)alkanoyloxymethyl esters such aspivaloyloxymethyl esters, 3-phthalidyl esters,(3-8C)cycloalkylcarbonyloxy-(1-6C)alkyl esters such ascyclopentylcarbonyloxymethyl and 1-cyclohexylcarbonyloxyethyl esters,2-oxo-1,3-dioxolenylmethyl esters such as5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl esters and(1-6C)alkoxycarbonyloxy-(1-6C)alkyl esters such asmethoxycarbonyloxymethyl and 1-methoxycarbonyloxyethyl esters.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula I which have a carboxy group is for example an in vivo cleavableamide such as a N—C₁₋₆alkyl and N,N-di-(C₁₋₆alkyl)amide such asN-methyl, N-ethyl, N-propyl, N,N-dimethyl, N-ethyl-N-methyl orN,N-diethylamide.

The in vivo effects of a compound of the Formula (I) may be exerted inpart by one or more metabolites that are formed within the human oranimal body after administration of a compound of the Formula (I). Asstated hereinbefore, the in vivo effects of a compound of the Formula(I) may also be exerted by way of metabolism of a precursor compound (apro-drug).

Two isomeric active metabolites of Example 1 have been identified fromin-vitro human systems as shown below (where the isomers arediastereomeric as a result of both configurations existing at the carbonmarked with a *), and synthesis of both isomers is set out in Examples14A and B herein:

Additionally, the following compound is believed to be an activemetabolite in some species, such as in mouse:

Such active metabolites form further independent aspects of theinvention.

For the avoidance of doubt it is to be understood that where in thisspecification a group is qualified by ‘hereinbefore defined’ or ‘definedhereinbefore’ the said group encompasses the first occurring andbroadest definition as well as each and all of the particulardefinitions for that group.

Particular novel compounds of the invention include, for example,compounds of the Formula (I), or pharmaceutically-acceptable saltsthereof, wherein, unless otherwise stated, each of R¹ and R², has any ofthe meanings defined hereinbefore or in the following statements:

In one aspect R¹ is hydrogen. In another aspect R¹ is fluoro.

In one aspect R² is hydrogen. In another aspect R² is fluoro.

In one aspect both R¹ and R² are hydrogen. In another aspect both R¹ andR² are fluoro. In another aspect R¹ is hydrogen and R² is fluoro.

In one aspect R³ is hydrogen. In another aspect R³ is methyl.

In one aspect R⁴ is hydrogen and R⁵ is fluoro. In another aspect R⁵ ishydrogen and R⁴ is fluoro.

Particular compounds of the invention are, for example, the compounds ofthe Formula (I) that are disclosed within the Examples that are set outhereinafter.

For example, a particular compound of the invention is a compound of theFormula (I) selected from any one of the following:—

-   (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   or a pharmaceutically-acceptable salt thereof.

A further particular compound of the invention is a compound of theFormula (I) selected from any one of the following:—

-   (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3,5-difluoro-4(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3,5-difluoro-4(1R)-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(4(1R)-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid;-   (E)-3-(3-fluoro-4(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic    acid; and-   (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoic    acid;-   or a pharmaceutically-acceptable salt thereof.

A particular pharmaceutically-acceptable salt of the invention is(1R,3R)-1-{4-[(E)-2-carboxyethenyl]-2,6-difluorophenyl}-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-beta-carbolin-2-iummaleate.

Another aspect of the present invention provides a process for preparinga compound of the Formula (I), or a pharmaceutically-acceptable saltthereof. A suitable process is illustrated by the followingrepresentative process variants in which, unless otherwise stated, R¹ toR⁵ have any of the meanings defined hereinbefore. Necessary startingmaterials may be obtained by standard procedures of organic chemistry.The preparation of such starting materials is described in conjunctionwith the following representative process variants and within theaccompanying Examples. Alternatively, necessary starting materials areobtainable by analogous procedures to those illustrated which are withinthe ordinary skill of an organic chemist.

Compounds of formula (I) are conveniently made by hydrolysis of an esterderivative of formula (II), wherein R⁶ is (1-6C) alkyl, such as methyl.Hydrolysis is conveniently carried out in the presence of base such asusing sodium hydroxide in a suitable solvent (such as aqueous THF andMeOH (or another similar alcohol), or such as an aqueous alcohol forexample aqueous isopropanol) and a suitable temperature, convenientlyroom temperature.

Compounds of formula (II) may be made by, for example:

a) reaction of a compound of formula (III) with a compound of formula(IV) under conditions known in the art as suitable for Pictet-Spenglerreactions (such as in the presence of acid (such as acetic acid) and ina suitable solvent (for example toluene) and a suitable temperature(such as 80° C.)); or

b) by reaction of a compound of formula (V) with a compound of formula(VI), where LG is a leaving group known in the art such as halide ortrifluoromethanesulfonate (triflate), conveniently triflate, in thepresence of base (for example an amine base such asN-ethyl-N-isopropylpropan-2-amine) and a suitable polar solvent (such asdioxane) at a suitable temperature (such as from room temperature to 90°C.).

Compounds of formula (III) may be prepared by reaction of a compound offormula (VII) with a compound of formula (VI) under conditions asdescribed for the reaction of compounds of formulae (V) and (VI) above.

Compounds of formula (IV) may be prepared by reaction of a compound offormula (VIII) with an alkyl acrylate ester (such as methyl acrylatewhen R⁶ is methyl) under conditions known in the art for a Heckreaction; that is in the presence of an aryl phosphine (egtri-o-tolylphosphine), a palladium catalyst (such as palladium (II)acetate and base (such as triethylamine) in a suitable solvent (such asDMA) and at a suitable temperature (eg 80° C.).

Compounds of formula (V) may be prepared by reaction of a compound offormula (VII) with a compound of formula (IV) using conditions similarto those described for the reaction of compounds of formulae (III) and(IV) above.

Compounds of formula (VI) where LG is triflate may be prepared as shownbelow in Schemes 1 and 2. Other compound of formula (VI) where LG isother than triflate may be prepared by similar methods known in the art.

Step 1: Fluorinating agent, e.g.N,N-diethyl-1,1,2,3,3,3-hexafluoropropan-1-amine/DCM/RT, then reducingagent, e.g. lithium aluminium hydride/THF/RTStep 2: Trifluoromethanesulfonic anhydride/base, e.g.2,6-lutidine/DCM/0° C.

Step 1: Reducing agent, e.g. lithium aluminium hydride/ether/0° C.Step 2: Trifluoromethanesulfonic anhydride/base, e.g.2,6-lutidine/DCM/−10° C.

Compounds of formula (I) are chiral. It will be understood by theskilled person that stereoselective reactions may be used to obtain thedesired isomers. Alternatively, stereochemistry may be adjusted bysuitable means, such as by epimerisation from cis to trans isomers viaacidification of an intermediate with protected amine group asillustrated in Example 4 herein (and described for example in J. Org.Chem. 2009, 74, 2771-2779).

In a further aspect of the invention there is provided a process formaking a compound of formula (I) comprising hydrolysis of a compound offormula (II), conveniently in the presence of base.

It is to be understood that other permutations of the process steps inthe process variants described above are also possible.

It is to be understood that any compound of Formula (I) obtained by anyof the processes described hereinbefore can be converted into anothercompound of the Formula (I) if required.

When a pharmaceutically-acceptable salt of a compound of the Formula (I)is required it may be obtained by, for example, reaction of saidcompound with a suitable base.

When a pharmaceutically-acceptable pro-drug of a compound of the Formula(I) is required, it may be obtained using a conventional procedure. Forexample, an in vivo cleavable ester of compound of the Formula (I) maybe obtained by, for example, reaction of a compound of the Formula (I)containing a carboxy group with a pharmaceutically-acceptable alcohol.Further information on pro-drugs has been provided hereinbefore.

It will also be appreciated that, in some of the reactions mentionedhereinbefore, it may be necessary or desirable to protect any sensitivegroups in the compounds. The instances where protection is necessary ordesirable, and suitable methods for protection, are known to thoseskilled in the art. Conventional protecting groups may be used inaccordance with standard practice (for illustration see T.W. Green,Protective Groups in Organic Synthesis, John Wiley and Sons, 1991).Thus, if reactants include groups such as amino, carboxy or hydroxy, itmay be desirable to protect the group in some of the reactions mentionedherein.

A suitable protecting group for an amino or alkylamino group is, forexample, an acyl group, for example an alkanoyl group such as acetyl, analkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl ort-butoxycarbonyl group, an arylmethoxycarbonyl group, for examplebenzyloxycarbonyl, or an aroyl group, for example benzoyl. Thedeprotection conditions for the above protecting groups necessarily varywith the choice of protecting group. Thus, for example, an acyl groupsuch as an alkanoyl or alkoxycarbonyl group or an aroyl group may beremoved for example, by hydrolysis with a suitable base such as analkali metal hydroxide, for example lithium or sodium hydroxide.Alternatively an acyl group such as a t-butoxycarbonyl group may beremoved, for example, by treatment with a suitable acid as hydrochloric,sulphuric or phosphoric acid or trifluoroacetic acid and anarylmethoxycarbonyl group such as a benzyloxycarbonyl group may beremoved, for example, by hydrogenation over a catalyst such aspalladium-on-carbon, or by treatment with a Lewis acid for example borontris(trifluoroacetate). A suitable alternative protecting group for aprimary amino group is, for example, a phthaloyl group which may beremoved by treatment with an alkylamine, for exampledimethylaminopropylamine, or with hydrazine.

A suitable protecting group for a hydroxy group is, for example, an acylgroup, for example an alkanoyl group such as acetyl, an aroyl group, forexample benzoyl, or an arylmethyl group, for example benzyl. Thedeprotection conditions for the above protecting groups will necessarilyvary with the choice of protecting group. Thus, for example, an acylgroup such as an alkanoyl or an aroyl group may be removed, for example,by hydrolysis with a suitable base such as an alkali metal hydroxide,for example lithium or sodium hydroxide. Alternatively an arylmethylgroup such as a benzyl group may be removed, for example, byhydrogenation over a catalyst such as palladium-on-carbon.

A suitable protecting group for a carboxy group is, for example, anesterifying group, for example a methyl or an ethyl group which may beremoved, for example, by hydrolysis with a base such as sodiumhydroxide, or for example a t-butyl group which may be removed, forexample, by treatment with an acid, for example an organic acid such astrifluoroacetic acid, or for example a benzyl group which may beremoved, for example, by hydrogenation over a catalyst such aspalladium-on-carbon.

The protecting groups may be removed at any convenient stage in thesynthesis using conventional techniques well known in the chemical art.

Certain of the intermediates (for example, compounds of the Formulae II,III, IV, V, VI, VII and VIII, particularly compounds of formula II, IIIand/or V) defined herein are novel and these are provided as a furtherfeature of the invention.

Biological Assays—

The following assays were used to measure the effects of the compoundsof the present invention.

ERα Binding Assay

The ability of compounds to bind to isolated Estrogen Receptor AlphaLigand binding domain (ER alpha—LBD (GST)) was assessed in competitionassays using a LanthaScreen™ Time-Resolved Fluorescence Resonance EnergyTransfer (TR-FRET) detection end-point. For the LanthaScreen TR-FRETendpoint, a suitable fluorophore (Fluormone ES2, Product code P2645) andrecombinant human Estrogen Receptor alpha ligand binding domain (Productcode PV4543) were purchased from Invitrogen and used to measure compoundbinding. The assay principle is that ER alpha-LBD (GST) is added to afluorescent ligand to form a receptor/fluorophore complex. Aterbium-labelled anti-GST antibody (Product code PV3551) is used toindirectly label the receptor by binding to its GST tag, and competitivebinding is detected by a test compounds' ability to displace thefluorescent ligand resulting in a loss of TR-FRET signal between theTb-anti-GST antibody and the tracer. The assay was performed as followswith all reagent additions carried out using the Beckman CoulterBioRAPTR FRD microfluidic workstation:—

-   -   1. Acoustic dispense 120 nl of the test compound into a black        low volume 384 well assay plates.    -   2. Prepare 1×ER alpha-LBD/Tb-antiGST Ab in ES2 screening buffer        and incubate for 20 minutes.    -   3. Add 1× fluorophore to the ER alpha-LBD/Tb-antiGST Ab solution        prior to use.    -   4. Dispense 12 μl of the 1×AR-LBD/Tb-anti-GST Ab/Fluorophore        reagent into each well of the assay plate    -   5. Cover the assay plate to protect the reagents from light and        evaporation, and incubate at room temperature for 1 hour.    -   6. Excite at 337 nm and measure the fluorescent emission signal        of each well at 490 nm and 520 nm using the BMG PheraSTAR.

Compounds were dosed directly from a compound source microplatecontaining serially diluted compound (4 wells containing 10 mM, 0.1 mM,1 μM and 10 nM final compound respectively) to an assay microplate usingthe Labcyte Echo 550. The Echo 550 is a liquid handler that usesacoustic technology to perform direct microplate-to-microplate transfersof DMSO compound solutions and the system can be programmed to transfermultiple small nL volumes of compound from the different source platewells to give the desired serial dilution of compound in the assay whichis then back-filled to normalise the DMSO concentration across thedilution range. In total 120 nL of compound plus DMSO is added to eachwell and compounds were tested in a 12-point concentration responseformat over a final compound concentration range of 100, 29.17, 10.42,2.083, 1, 0.292, 0.104, 0.02083, 0.01, 0.002917, 0.001042, 0.0001 μM,respectively. TR-FRET dose response data obtained with each compound wasexported into a suitable software package (such as Origin or Genedata)to perform curve fitting analysis. Competitive ER alpha binding wasexpressed as an IC₅₀ value. This was determined by calculation of theconcentration of compound that was required to give a 50% reduction intracer compound binding to ER alpha-LBD.

MCF-7 ER Down Regulation Assay

The ability of compounds to down-regulate Estrogen Receptor (ER) numberswas assessed in a cell based immuno-fluorescence assay using the MCF-7human ductal carcinoma breast cell line. MCF-7 cells were reviveddirectly from a cryovial (approx 5×10⁶ cells) in Assay Medium (phenolred free Dulbecco's Modified Eagle's medium (DMEM) (Sigma D5921)containing 2 mM L-Glutamine and 5% (v/v) Charcoal/Dextran treated foetalcalf serum Cells were syringed once using a sterile 18 G×1.5 inch(1.2×40 mm) broad gauge needle and cell density was measured using aCoulter Counter (Beckman). Cells were further diluted in Assay Medium toa density of 3.75×10⁴ cells per ml and 40 μl per well added totransparent bottomed, black, tissue culture treated 384 well plates(Costar, No. 3712) using a Thermo Scientific Matrix WellMate or ThermoMultidrop. Following cell seeding, plates were incubated overnight at37° C., 5% CO₂ (Liconic carousel incubator). Test data was generatedusing the LabCyte Echo® model 555 compound reformatter which is part ofan automated workcell (Integrated Echo 2 workcell). 10 mM compound stocksolutions of the test compounds were used to generate a 384 wellcompound dosing plate (Labcyte P-05525-CV1). 40 μl of each of the 10 mMcompound stock solutions was dispensed into the first quadrant well andthen 1:100 step-wise serial dilutions in DMSO were performed using aHydra II (MATRIX UK) liquid handling unit to give 40 ul of dilutedcompound into quadrant wells 2 (0.1 mM), 3 (1 μM) and 4 (0.01 μM),respectively. 40 μl of DMSO added to wells in row P on the source plateallow for DMSO normalisation across the dose range. To dose the controlwells 40 μl of DMSO was added to row O1 and 40 μl of 100 μM Faslodex® inDMSO was added to row O3 on the compound source plate. The Echo usesacoustic technology to perform direct microplate-to-microplate transfersof DMSO compound solutions to assay plates. The system can be programmedto transfer volumes as low as 2.5 nL in multiple increments betweenmicroplates and in so doing generates a serial dilution of compound inthe assay plate which is then back-filled to normalise the DMSOconcentration across the dilution range. Compounds were dispensed ontothe cell plates with a compound source plate prepared as above producinga 12 pt duplicate 3 μM to 3 pM dose range with 3 fold dilutions and onefinal 10 fold dilution using the Integrated Echo 2 workcell. The maximumsignal control wells were dosed with DMSO to give a final concentrationof 0.3% and the minimum signal control wells were dosed with Faslodex®to give a final concentration of 100 nM accordingly. Plates were furtherincubated for 18-22 hours at 37° C., 5% CO₂ and then fixed by theaddition of 20 μl of 11.1% (v/v) formaldehyde solution (in phosphatebuffered saline (PBS)) giving a final formaldehyde concentration of 3.7%(v/v). Cells were fixed at room temperature for 20 mins before beingwashed two times with 250 μl PBS/Proclin (PBS with a Biocidepreservative) using a BioTek platewasher, 40 μl of PBS/Proclin was thenadded to all wells and the plates stored at 4° C. The fixing methoddescribed above was carried out on the Integrated Echo 2 workcell.Immunostaining was performed using an automated AutoElisa workcell. ThePBS/Proclin was aspirated from all wells and the cells permeabilisedwith 40 μl PBS containing 0.5% Tween™ 20 (v/v) for 1 hour at roomtemperature. The plates were washed three times in 250 μl of PBS/0.05%(v/v) Tween 20 with Proclin (PBST with a Biocide preservative) and then20 μl of ERα (SP1) Rabbit monoclonal antibody (Thermofisher) 1:1000 inPBS/Tween™/3% (w/v) Bovine Serum Albumin was added. The plates wereincubated overnight at 4° C. (Liconic carousel incubator) and thenwashed three times in 250 μl of PBS/0.05% (v/v) Tween™ 20 with Proclin(PBST). The plates were then incubated with 20 μl/well of a goatanti-rabbit IgG AlexaFluor 594 or goat anti-rabbit AlexaFluor 488antibody (Molecular Probes) with Hoechst at 1:5000 in PBS/Tween™/3%(w/v) Bovine Serum Albumin for 1 hr at room temperature. The plates werethen washed three times in 250 μl of PBS/0.05% (v/v) Tween™ 20 withProclin (PBST with a Biocide preservative). 20 μl of PBS was added toeach well and the plates covered with a black plate seal and stored at4° C. before being read. Plates were read using a Cellomics Arrayscanreading the 594 nm (24 hr time point) or 488 nm (5 hr timepoint)fluorescence to measure the ERα receptor level in each well. The meantotal intensity was normalized for cell number giving the totalintensity per cell. The data was exported into a suitable softwarepackage (such as Origin) to perform curve fitting analysis.Down-regulation of the ERα receptor was expressed as an IC₅₀ value andwas determined by calculation of the concentration of compound that wasrequired to give a 50% reduction of the average maximum Total Intensitysignal.

Although the pharmacological properties of the compounds of the Formula(I) vary with structural change as expected, in general activitypossessed by compounds of the Formula (I) may be demonstrated at thefollowing concentrations or doses in one or more of the above tests.

The following data were generated for the Examples (the data below maybe a result from a single experiment or an average of multiple repeatexperiments):

TABLE A Example ER binding IC₅₀ value ER down regulation IC₅₀ value 1<0.64 0.14 2 1 0.85 3 1 0.4 4 1.6 0.99 5 0.2 0.57 6 <1.3 0.44 7 5 1.7 82.2 3 9 <1.2 1.5MCF-7 In-vivo Xenograft Study with Example 1 as Single Agent and inCombination with an mTOR Inhibitor.

MCF7 cells (5×10⁶ cells suspended in 100 μl of RPMI cell medium) wereimplanted subcutaneously in the hind flank of immuno-compromised (SCID)mice the day after each mouse was surgically implanted with a 0.5 mg/21day oestrogen pellet (Innovative Research, USA). Tumours were measuredtwice weekly and changes in tumour volume and growth inhibition weredetermined by bilateral Vernier calliper measurement (length×width)where length was taken to be the longest diameter across the tumour andwidth the corresponding perpendicular. Tumour volume was calculatedusing the formula (length×width)×√(length×width)×π/6).

Tumours were measured 13 days after cell implantation to allowrandomisation of mice into test groups. Treatment with compounds beganthe day after (i.e. 14 days after cell implantation).

The mTOR inhibitor AZD2014 was dosed to different groups of mice at 15mg/kg once daily every day orally (p.o.) at a volume of 0.1 ml per 10 g.Example 1 was dosed at 5 mg/kg once daily orally at 0.1 ml/10 g. Onegroup of animals was dosed with vehicle p.o. to act as a control. Ninemice per group were used for active agents for the control group.

The data obtained from this study are shown in FIG. 10.

The effect of a combination of a compound of Formula (I) with aninhibitor of PI3Kα/δ may be studied in a similar manner to thecombination with the mTOR inhibitor above.

HCC1428 Long Term Estrogen Deprived (HCC1428 LTED) Xenograft EfficacyStudy

After a suitable period of cell culture, HCC1428 LTED cells (1×10⁶) wereimplanted subcutaneously in the hind flank of female immuno-compromisedNSG mice (Jackson Labs, USA) that had undergone overectomy. Tumours weremeasured twice weekly and changes in tumour volume and growth inhibitionwere determined by bilateral Vernier calliper measurement (length×width)where length was taken to be the longest diameter across the tumour andwidth the corresponding perpendicular. Tumour volume was calculatedusing the formula (length×width)×√(length×width)×(π/6). Tumours weremeasured once weekly after cell implantation until the average sizereached 150 mm³ at which point the mice were placed into randomised testgroups, each group containing 10 mice. Treatment with compounds beganthe day after (day 62 in this study) and once weekly tumour measurementcontinued. Example 1 was dosed 25 mg/kg once daily every day orally(p.o.) at a volume of 0.1 ml per 10 g. Another group of animals wasdosed with vehicle p.o. to act as a control.

After 28 days of dosing, control treated tumours grew on average by 220mm³ (using geometric mean values) while tumours from mice treated byExample 1 decreased in size by 46 mm³ representing a 121% inhibition oftumour growth (P<0.001 by unpaired t-test).

To measure the levels of estrogen receptor protein in xenograft tumours,tumours samples were harvested 24 hrs post final dose of vehicle orexample 1 treatment and snap frozen in liquid nitrogen. For proteinextraction, tumour fragments were added to 700 ul of Invitrogen CellExtraction buffer (FNN0011) with added Sigma Phosphatase inhibitors (No.2 (P5726) and 3 (P0044) 1 in 100 dilution) and Roche Complete(11836145001) protease inhibitor (1 tablet per 50 mls), 1 mMdithiothreitol (DTT) in 2 ml sample tubes on wt ice. Homogenisation ofthe sample was done using a Mixermill (level 27/sec) and 3×2 mins cyclesof homogenisation. Samples were spun briefly to ascertain completehomogenisation of the tumours. Homogenate was sonicated for 10 secondsand then spun down at top speed (13000 rpm) centrifuge for 15 mins.Levels of protein in the supernatent were measured and approx 45 ug ofprotein were run on a 15 well Bis-Tris Gels (4-12% Gels) using standardmethods. Following protein separation and transfer onto nitrocellulosefilter, estrogen receptor 68 kDa: ThermoFisher SP1 #9101S antibody wasadded, diluted 1:400 in milk/PBS/T and incubated overnight at 4° C. Thefilter was washed in 3×5 mins in ˜20 ml of TBS/T 0.05% and a secondaryanti-rabbit detection antibody was diluted 1:2000 in 5% marvel in TBS/Tand incubate for 1 hr at RT. Signal was detected using chemiluminescentSuperSignal West Dura extended Duration substrate and quantified usingSyngene software. Vinculin protein levels were measured as a loadingcontrol using V931 Sigma diluted 1:10,000 in marvel and an anti-mousedetection antibody. The results in FIGS. 11 and 12 show that a 60%decrease in ER levels were observed upon treatment with Example 1relative to vehicle control.

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a compound of the Formula(I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore in association with a pharmaceutically-acceptable diluentor carrier.

Suitable pharmaceutically-acceptable excipients for a tablet formulationinclude, for example, inert diluents, granulating and disintegratingagents, binding agents, lubricating agents, preservative agents andanti-oxidants. A further suitable pharmaceutically-acceptable excipientmay be a chelating-agent. Tablet formulations may be uncoated or coatedeither to modify their disintegration and the subsequent absorption ofthe active ingredient within the gastrointestinal tract, or to improvetheir stability and/or appearance, in either case, using conventionalcoating agents and procedures well known in the art.

Compositions for oral use may alternatively be in the form of hardgelatin capsules in which the active ingredient is mixed with an inertsolid diluent, or as soft gelatin capsules in which the activeingredient is mixed with water or an oil.

Aqueous suspensions generally contain the active ingredient in finelypowdered form together with one or more suspending agents, dispersing orwetting agents. The aqueous suspensions may also contain one or morepreservatives, anti-oxidants, colouring agents, flavouring agents,and/or sweetening agents.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil or in a mineral oil. The oily suspensions may alsocontain a thickening agent. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Additional excipients such as sweetening,flavouring and colouring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil or amineral oil or a mixture of any of these. The emulsions may also containsweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents, and mayalso contain a demulcent, preservative, flavouring and/or colouringagent.

The pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oily suspension, which may be formulated accordingto known procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solventsystem.

Compositions for administration by inhalation may be in the form of aconventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient. Dry powder inhalers may also be suitable.

For further information on formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990.

In one aspect of the invention, the pharmaceutical composition describedabove comprises the compound of Example 1[(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid], or a pharmaceutically acceptable salt thereof. Conveniently, thecompound of Example 1 is present in its polymorph described herein ascrystalline Form B.

The process for synthesising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid as set out in Example 1, recommends that the process is carried outin the absence of light and under a nitrogen atmosphere in order toavoid the formation of a degradation product.

The degradation product referred to, which is(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate,has the following structure:

and is thought may be formed from Example 1[(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid] by autooxidation in air via a free radical chain mechanism. Forthe avoidance of doubt, this degradation product is not believed to havesignificant SERD activity.

This compound could also be referred to as(E)-3-[3,5-difluoro-4-[(3R)-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoateand a synthetic method for making it is given in Example 13.

The skilled person will appreciate that control of the formation ofdegradation products is essential to the safe production and storage ofpharmaceuticals. Also, the skilled person will appreciate that certaincompounds may degrade on storage, even after formulation into apharmaceutical composition, and that such degradation may in someinstances be controlled by the use of appropriate excipients in thepharmaceutical composition and/or by appropriate packaging of the finalproduct. The skilled person will further appreciate that a finalformulation which is developed for commercial use will need to beoptimised for a number of characteristics, including chemical stability,but also including for instance physical stability and dissolutioncharacteristics. Therefore such formulations will be developed in orderto balance a number of different factors.

Suitably, compositions of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid, or a pharmaceutically-acceptable salt thereof, include a compoundwhich acts as an anti-oxidant.

Anti-oxidant compounds which are suitable for use in pharmaceuticalcompositions are known in the art, and include, for example, acetonesodium bisulfite; alpha lipoic acid; alpha tocopherol; ascorbic acid;ascorbyl palmitate; butylated hydroxyanisole; butylated hydroxytoluene;carotenes; citric acid monohydrate; dodecyl gallate; erythorbic acid;fumaric acid; glutathione; histidine; hypophosphorous acid; lactobionicacid; lipoic acid; malic acid; melatonin; methionine; d-mannose;monothioglycerol; octyl gallate; potassium metabisulfite; propionicacid; propyl gallate; sodium ascorbate; sodium bisulfite; sodiumformaldehyde sulfoxylate; sodium metabisulfite; sodium sulfite; sodiumthiosulfate; stannous chloride; sulfur dioxide; thymol; tocopherol;tocotrienols; ubiquinol; uric acid; vitamin E; and vitamin Epolyethylene glycol succinate. Such compounds may exert theiranti-oxidant effect by a variety of mechanisms, and one or more of thesemechanisms may be more effective than others for any particularcompound. Some anti-oxidants compounds, such as BHA, act as free-radicalscavengers. Other anti-oxidants, such sodium metabisulfite and ascorbicacid are easily oxidised and so may be oxidised in preference to theactive ingredient.

For example, where metal induced peroxide formation is involved in theoxidation mechanism, use of a chelating agent, such as for example EDTA(ethylenediaminetetraacetic acid), may be useful to remove any metalcontaminants and thereby indirectly achieve a stabilising effect. Othermetal-chelating agents are known in the art and include, for example,betadex sulfobutyl ether sodium; calcium acetate; citric acidmonohydrate; cyclodextrins; disodium edetate; edetic acid; fumaric acid;galactose; glutamic acid; histidine; hydroxypropyl betadex; malic acid;pentetic acid; phytochelatin; poly(methyl vinyl ether/maleic anhydride);potassium citrate; sodium citrate dihydrate; sodium phosphate, dibasic;sodium phosphate, monobasic; tartaric acid; and trehalose.

For example, propyl gallate, sodium metabisulfite, ascorbic acid andbutylated hydroxyanisole were included in exemplary formulations ofExample 1. EDTA was also included. An example of such a formulation isprovided as Example 12. Of these, compositions containing sodiummetabisulfite appeared to be less stable than those with noanti-oxidant, after four weeks storage under a number of different heatand humidity conditions. Compositions containing ascorbic acid appearedto be the most stable after 4 weeks storage under a number of differentheat and humidity conditions (as determined by Liquid Chromatographyanalysis, eg UHPLC).

Further suitable additives for formulations comprising the compound ofExample 1, include using excipients with a low metal content, excipientswith a low peroxide content, excipients such as mannitol which is afree-radical scavenger as well as a filler. The process of production ofsuch a formulation may also impact stability. For example, for someactive ingredients, ensuring intimate mixing of the active ingredientwith stability-inducing excipients may be important in ensuring maximumstabilisation. Intimate mixing may be influenced by, for example, mixingspeed, particle sizes and wet or dry mixing/granulation processes. Anactive ingredient may be granulated with an antioxidant and then mixedwith other excipients. Antioxidants may also be added to any coating onthe outside of a pharmaceutical composition.

In one aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluents or carrier.

In one aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, and also comprisingan anti-oxidant. Suitably, ascorbic acid may be used as theanti-oxidant.

In one aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, and also comprisinga metal chelating agent. Suitably, EDTA may be used as a metal chelatingagent.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, an anti-oxidant andoptionally further comprising a metal chelating agent.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluent or carrier, wherein thecomposition contains less than 5% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluent or carrier, wherein thecomposition contains less than 2% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluent or carrier, wherein thecomposition contains less than 1% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluent or carrier, wherein thecomposition contains less than 0.5% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluents or carrier, whereinthe composition contains less than 0.1% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically-acceptable salt thereof, in association withat least one pharmaceutically-acceptable diluents or carrier, whereinthe composition contains less than 0.05% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

In the above aspects, where the composition is described as containingless than 5% w/w, 2% w/w, 1% w/w, 0.5% w/w, 0.1% w/w or 0.05% w/w of(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylatethen the skilled person will understand that this is intended to meanpercentage weight for weight in comparison to the weight of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid present in the composition.

The degradation product(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylatemay therefore be used as a reference marker or reference standard inanalytical techniques such as HPLC to monitor the stability of thecompound of Example 1 or pharmaceutical compositions containing it.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, oral administration to humans willgenerally require, for example, from 1 mg to 2 g of active agent (moresuitably from 100 mg to 2 g, for example from 250 mg to 1.8 g, such asfrom 500 mg to 1.8 g, particularly from 500 mg to 1.5 g, convenientlyfrom 500 mg to 1 g) to be administered compounded with an appropriateand convenient amount of excipients which may vary from about 3 to about98 percent by weight of the total composition. It will be understoodthat, if a large dosage is required, multiple dosage forms may berequired, for example two or more tablets or capsules, with the dose ofactive ingredient divided conveniently between them. Typically, unitdosage forms will contain about 10 mg to 0.5 g of a compound of thisinvention, although a unit dosage form may contain up to 1 g.Conveniently, a single solid dosage form may contain between 1 and 300mg of active ingredient.

The size of the dose for therapeutic or prophylactic purposes ofcompounds of the present invention will naturally vary according to thenature and severity of the disease state, the age and sex of the animalor patient and the route of administration, according to well knownprinciples of medicine.

In using compounds of the present invention for therapeutic orprophylactic purposes it will generally be administered so that a dailydose in the range, for example, 1 mg/kg to 100 mg/kg body weight isreceived, given if required in divided doses. In general, lower doseswill be administered when a parenteral route is employed. Thus, forexample, for intravenous administration, a dose in the range, forexample, 1 mg/kg to 25 mg/kg body weight will generally be used.Similarly, for administration by inhalation, a dose in the range, forexample, 1 mg/kg to 25 mg/kg body weight will be used. Oraladministration is however preferred, particularly in tablet form.

In one aspect of the invention, compounds of the present invention orpharmaceutically-acceptable salts thereof, are administered as tabletscomprising 10 mg to 100 mg of the compound of Formula (I) (or apharmaceutically-acceptable salt thereof), wherein one or more tabletsare administered as required to achieve the desired dose.

As stated above, it is known that signalling through ERα causestumourigenesis by one or more of the effects of mediating proliferationof cancer and other cells, mediating angiogenic events and mediating themotility, migration and invasiveness of cancer cells. We have found thatthe compounds of the present invention possess potent anti-tumouractivity which it is believed is obtained by way of antagonism anddown-regulation of ERα that is involved in the signal transduction stepswhich lead to the proliferation and survival of tumour cells and theinvasiveness and migratory ability of metastasising tumour cells.

Accordingly, the compounds of the present invention may be of value asanti-tumour agents, in particular as selective inhibitors of theproliferation, survival, motility, dissemination and invasiveness ofmammalian cancer cells leading to inhibition of tumour growth andsurvival and to inhibition of metastatic tumour growth. Particularly,the compounds of the present invention may be of value asanti-proliferative and anti-invasive agents in the containment and/ortreatment of solid tumour disease. Particularly, the compounds of thepresent invention may be useful in the prevention or treatment of thosetumours which are sensitive to inhibition of ERα and that are involvedin the signal transduction steps which lead to the proliferation andsurvival of tumour cells and the migratory ability and invasiveness ofmetastasising tumour cells. Further, the compounds of the presentinvention may be useful in the prevention or treatment of those tumourswhich are mediated alone or in part by antagonism and down-regulation ofERα, i.e. the compounds may be used to produce an ERα inhibitory effectin a warm blooded animal in need of such treatment.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use as a medicament in awarm-blooded animal such as man.

According to a further aspect of the invention, there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in the production of ananti-proliferative effect in a warm-blooded animal such as man.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in a warm-blooded animal suchas man as an anti-invasive agent in the containment and/or treatment ofsolid tumour disease.

According to a further aspect of the invention, there is provided theuse of a compound of the Formula (I), or a pharmaceutically-acceptablesalt thereof, as defined hereinbefore for the production of ananti-proliferative effect in a warm-blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the production of an anti-proliferative effect in a warm-bloodedanimal such as man.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in a warm-blooded animal such as man as an anti-invasive agent inthe containment and/or treatment of solid tumour disease.

According to a further aspect of the invention there is provided amethod for producing an anti-proliferative effect in a warm bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention there is provided amethod for producing an anti-invasive effect by the containment and/ortreatment of solid tumour disease in a warm blooded animal, such as man,in need of such treatment which comprises administering to said animalan effective amount of a compound of the Formula (I), or apharmaceutically-acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention, there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in the prevention or treatmentof cancer in a warm blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the prevention or treatment of cancer in a warm blooded animalsuch as man.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of cancer in a warm bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention, there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in the prevention or treatmentof solid tumour disease in a warm blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the prevention or treatment of solid tumour disease in a warmblooded animal such as man.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of solid tumour disease in a warmblooded animal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in the prevention or treatmentof those tumours which are sensitive to inhibition of ERα that areinvolved in the signal transduction steps which lead to theproliferation, survival, invasiveness and migratory ability of tumourcells.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the prevention or treatment of those tumours which are sensitiveto inhibition of ERα that are involved in the signal transduction stepswhich lead to the proliferation, survival, invasiveness and migratoryability of tumour cells.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of those tumours which aresensitive to inhibition of ERα that are involved in the signaltransduction steps which lead to the proliferation, survival,invasiveness and migratory ability of tumour cells which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in providing an inhibitoryeffect on ERα.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in providing an inhibitory effect on ERα.

According to a further aspect of the invention there is also provided amethod for providing an inhibitory effect on ERα which comprisesadministering an effective amount of a compound of the Formula (I), or apharmaceutically-acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore for use in providing a selectiveinhibitory effect on ERα.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in providing a selective inhibitory effect on ERα.

According to a further aspect of the invention there is also provided amethod for providing a selective inhibitory effect on ERα whichcomprises administering an effective amount of a compound of the Formula(I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore.

Described herein are compounds that can bind to ERα ligand bindingdomain and are selective estrogen receptor degraders. In biochemical andcell based assays the compounds of the present invention are shown to bepotent estrogen receptor binders and reduce cellular levels of ERα andmay therefore be useful in the treatment of estrogen sensitive diseasesor conditions (including diseases that have developed resistance toendocrine therapies), i.e. for use in the treatment of cancer of thebreast and gynaecological cancers (including endometrial, ovarian andcervical) and cancers expressing ERα mutated proteins which may be denovo mutations or have arisen as a result of treatment with a priorendocrine therapy such as an aromatase inhibitor.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before for use in the treatment of breast orgynaecological cancers.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before for use in the treatment of cancer ofthe breast, endometrium, ovary or cervix.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before for use in the treatment of cancer ofthe breast.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before for use in the treatment of cancer ofthe breast, wherein the cancer has developed resistance to one or moreother endocrine therapies.

According to a further aspect of the invention there is provided amethod for treating breast or gynaecological cancers, which comprisesadministering an effective amount of a compound of the Formula (I), or apharmaceutically-acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention there is provided amethod for treating cancer of the breast, endometrium, ovary or cervix,which comprises administering an effective amount of a compound of theFormula (I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention there is provided amethod for treating breast cancer, which comprises administering aneffective amount of a compound of the Formula (I), or apharmaceutically-acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention there is provided amethod for treating breast cancer, wherein the cancer has developedresistance to one or more other endocrine therapies, which comprisesadministering an effective amount of a compound of the Formula (I), or apharmaceutically-acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before in the manufacture of a medicament foruse in the treatment of breast or gynaecological cancers.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before in the manufacture of a medicament foruse in the treatment of cancer of the breast, endometrium, ovary orcervix.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before in the manufacture of a medicament foruse in the treatment of breast cancer.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before in the manufacture of a medicament foruse in the treatment of breast cancer, wherein the cancer has developedresistance to one or more other endocrine therapies.

In one feature of the invention, the cancer to be treated is breastcancer. In a further aspect of this feature, the breast cancer isEstrogen Receptor+ve (ER+ve). In one embodiment of this aspect, thecompound of Formula (I) is dosed in combination with another anticanceragent, such as an anti-hormonal agent as defined herein.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before for use in the treatment of ER+vebreast cancer.

According to a further aspect of the invention there is provided amethod for treating ER+ve breast cancer, which comprises administeringan effective amount of a compound of the Formula (I), or apharmaceutically-acceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically-acceptable saltthereof, as defined herein before in the manufacture of a medicament foruse in the treatment of ER+ve breast cancer.

As stated hereinbefore, the in vivo effects of a compound of the Formula(I) may be exerted in part by one or more metabolites that are formedwithin the human or animal body after administration of a compound ofthe Formula (I).

The present invention therefore also contemplates a method forinhibiting ER-α in a patient, comprising administering to a patient anamount of a compound of Formula (I), or a pharmaceutically-acceptablesalt thereof, effective in inhibiting ER-α in the patient.

The present invention therefore also contemplates a method forinhibiting ER-α in a patient, comprising administering to a patient anamount of a compound of Formula (I), or a pharmaceutically-acceptablesalt thereof, effective in inhibiting ER-α in the patient.

In all of the above uses and methods, a suitable compound of the formula(I) is Example 1, or a pharmaceutically-acceptable salt thereof. In oneaspect, Example 1 is in crystalline form B as described herein.

The anti-cancer treatment defined herein may be applied as a soletherapy or may involve, in addition to the compounds of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti-tumouragents:—

-   (i) other antiproliferative/antineoplastic drugs and combinations    thereof, as used in medical oncology, such as alkylating agents (for    example cis-platin, oxaliplatin, carboplatin, cyclophosphamide,    nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide    and nitrosoureas); antimetabolites (for example gemcitabine and    antifolates such as fluoropyrimidines like 5-fluorouracil and    tegafur, raltitrexed, methotrexate, cytosine arabinoside, and    hydroxyurea); antitumour antibiotics (for example anthracyclines    like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,    idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic    agents (for example vinca alkaloids like vincristine, vinblastine,    vindesine and vinorelbine and taxoids like taxol and taxotere and    polokinase inhibitors); and topoisomerase inhibitors (for example    epipodophyllotoxins like etoposide and teniposide, amsacrine,    topotecan and camptothecin);-   (ii) antihormonal agents such as antioestrogens (for example    tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and    iodoxyfene), progestogens (for example megestrol acetate), aromatase    inhibitors (for example as anastrozole, letrozole, vorazole and    exemestane);-   (iii) inhibitors of growth factor function and their downstream    signalling pathways: included are Ab modulators of any growth factor    or growth factor receptor targets, reviewed by Stem et al. Critical    Reviews in Oncology/Haematology, 2005, 54, pp 11-29); also included    are small molecule inhibitors of such targets, for example kinase    inhibitors—examples include the anti-erbB2 antibody trastuzumab    [Herceptin™], the anti-EGFR antibody panitumumab, the anti-EGFR    antibody cetuximab [Erbitux, C225] and tyrosine kinase inhibitors    including inhibitors of the erbB receptor family, such as epidermal    growth factor family receptor (EGFR/erbB1) tyrosine kinase    inhibitors such as gefitinib or erlotinib, erbB2 tyrosine kinase    inhibitors such as lapatinib, and mixed erb1/2 inhibitors such as    afatanib; similar strategies are available for other classes of    growth factors and their receptors, for example inhibitors of the    hepatocyte growth factor family or their receptors including c-met    and ron; inhibitors of the insulin and insulin growth factor family    or their receptors (IGFR, IR) inhibitors of the platelet-derived    growth factor family or their receptors (PDGFR), and inhibitors of    signalling mediated by other receptor tyrosine kinases such as    c-kit, AnLK, and CSF-1R;-   also included are modulators which target signalling proteins in the    PI3-kinase signalling pathway, for example, inhibitors of PI3-kinase    isoforms such as PI3K-α/β/γ and ser/thr kinases such as AKT, mTOR    (such as AZD2014), PDK, SGK, PI4K or PIP5K; also included are    inhibitors of serine/threonine kinases not listed above, for example    raf inhibitors such as vemurafenib, MEK inhibitors such as    selumetinib (AZD6244), Abl inhibitors such as imatinib or nilotinib,    Btk inhibitors such as ibrutinib, Syk inhibitors such as    fostamatinib, aurora kinase inhibitors (for example AZD1152),    inhibitors of other ser/thr kinases such as JAKs, STATs and IRAK4,    and cyclin dependent kinase inhibitors such as palbociclib; iv)    modulators of DNA damage signalling pathways, for example PARP    inhibitors (e.g. Olaparib), ATR inhibitors or ATM inhibitors; v)    modulators of apoptotic and cell death pathways such as Bcl family    modulators (e.g. ABT-263/Navitoclax, ABT-199);-   (vi) antiangiogenic agents such as those which inhibit the effects    of vascular endothelial growth factor, [for example the    anti-vascular endothelial cell growth factor antibody bevacizumab    (Avastin™) and for example, a VEGF receptor tyrosine kinase    inhibitor such as sorafenib, axitinib, pazopanib, sunitinib and    vandetanib (and compounds that work by other mechanisms (for example    linomide, inhibitors of integrin αvβ3 function and angiostatin)];-   (vii) vascular damaging agents, such as Combretastatin A4;-   (viii) anti-invasion agents, for example c-Src kinase family    inhibitors like (dasatinib, J. Med. Chem., 2004, 47, 6658-6661) and    bosutinib (SKI-606), and metalloproteinase inhibitors like    marimastat, inhibitors of urokinase plasminogen activator receptor    function or antibodies to Heparanase];-   (ix) immunotherapy approaches, including for example ex-vivo and    in-vivo approaches to increase the immunogenicity of patient tumour    cells, such as transfection with cytokines such as interleukin 2,    interleukin 4 or granulocyte-macrophage colony stimulating factor,    approaches to decrease T-cell anergy, approaches using transfected    immune cells such as cytokine-transfected dendritic cells,    approaches using cytokine-transfected tumour cell lines and    approaches using anti-idiotypic antibodies. Specific examples    include monoclonal antibodies targeting PD-1 (e.g. BMS-936558) or    CTLA4 (e.g. ipilimumab and tremelimumab);-   (x) Antisense or RNAi based therapies, for example those which are    directed to the targets listed.-   (xi) gene therapy approaches, including for example approaches to    replace aberrant genes such as aberrant p53 or aberrant BRCA1 or    BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such    as those using cytosine deaminase, thymidine kinase or a bacterial    nitroreductase enzyme and approaches to increase patient tolerance    to chemotherapy or radiotherapy such as multi-drug resistance gene    therapy.

According to this aspect of the invention there is provided acombination suitable for use in the treatment of cancer comprisingcompounds of the present invention as defined herein or apharmaceutically acceptable salt thereof and another anti-tumour agent,in particular any one of the anti tumour agents listed under (i)-(xi)above. In particular, the anti-tumour agent listed under (i)-(xi) aboveis the standard of care for the specific cancer to be treated; theperson skilled in the art will understand the meaning of “standard ofcare”. In one aspect, the compound of the present invention is Example1, or a pharmaceutically-acceptable salt thereof.

Therefore in a further aspect of the invention there is providedcompounds of the present invention or a pharmaceutically acceptable saltthereof in combination with another anti-tumour agent, in particular ananti-tumour agent selected from one listed under (i)-(xi) herein above.

In a further aspect of the invention there is provided Example 1[(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid] or a pharmaceutically acceptable salt thereof in combination withanother anti-tumour agent, in particular an anti-tumour agent selectedfrom one listed under (i)-(xi) herein above.

In a further aspect of the invention there is provided compounds of thepresent invention or a pharmaceutically acceptable salt thereof incombination with another anti-tumour agent, in particular an anti-tumouragent selected from one listed under (i) above.

In a further aspect of the invention there is provided a compound of thepresent invention as defined herein before or a pharmaceuticallyacceptable salt thereof and any one of the anti tumour agents listedunder (i) above.

In a further aspect of the invention there is provided Example 1 or apharmaceutically acceptable salt thereof in combination with any one ofthe anti-tumour agents listed under (i) above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising compounds of thepresent invention as defined hereinbefore or a pharmaceuticallyacceptable salt thereof and a taxoid, such as for example taxol ortaxotere, conveniently taxotere. For example, a suitable compound of theinvention in combination with a taxoid, such as for example taxol ortaxotere, conveniently taxotere, is Example 1, or apharmaceutically-acceptable salt thereof.

In a further aspect of the invention there is provided compounds of thepresent invention or a pharmaceutically acceptable salt thereof incombination with another anti-tumour agent, in particular an anti-tumouragent selected from one listed under (ii) herein above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising compounds of thepresent invention as defined herein before or a pharmaceuticallyacceptable salt thereof and any one of antihormonal agents listed under(ii) above, for example any one of the anti-oestrogens listed in (ii)above, or for example an aromatase inhibitor listed in (ii) above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising Example 1 or apharmaceutically acceptable salt thereof and any one of antihormonalagents listed under (ii) above, for example any one of theanti-oestrogens listed in (ii) above, or for example an aromataseinhibitor listed in (ii) above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising Example 1 or apharmaceutically acceptable salt thereof and an mTOR inhibitor, such asAZD2014 (see for example WO2008/023161).

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising Example 1, or apharmaceutically-acceptable salt thereof and a PI3Kα-inhibitor, such asthose PI3K α/δ inhibitors in our co-pending PCT applicationPCT/GB2014/050163. One example of a suitable PI3K α/δ inhibitor isExample 3 from PCT/GB2014/050163, which is the compound1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,or a pharmaceutically-acceptable salt thereof. A process to make Example3 of PCT/GB2014/050163 is set out in Reference Example 1 herein.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising Example 1 or apharmaceutically acceptable salt thereof and palbociclib.

In one aspect the above combination of a compound of formula (I) or apharmaceutically acceptable salt thereof, particularly Example 1 or apharmaceutically acceptable salt thereof, with an anti-tumour agentlisted in (ii) above, or an mTOR inhibitor (such as AZD2014), or aPI3K-α inhibitor (such as those PI3K α/δ inhibitors in our co-pendingPCT application PCT/GB2014/050163, particularly Example 3 therein) orpalbociclib, is suitable for use in the treatment of breast orgynaecological cancers, such as cancer of the breast, endometrium, ovaryor cervix, particularly breast cancer, such as ER+ve breast cancer.

Herein, where the term “combination” is used it is to be understood thatthis refers to simultaneous, separate or sequential administration. Inone aspect of the invention “combination” refers to simultaneousadministration. In another aspect of the invention “combination” refersto separate administration. In a further aspect of the invention“combination” refers to sequential administration. Where theadministration is sequential or separate, the delay in administering thesecond component should not be such as to lose the beneficial effect ofthe combination. Where a combination of two or more components isadministered separately or sequential, it will be understood that thedosage regime for each component may be different to and independent ofthe other components. Conveniently, the compounds of the presentinvention are dosed once daily.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises Example 1[(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid] or a pharmaceutically acceptable salt thereof in combination withan anti-tumour agent selected from one listed under (i)-(xi) hereinabove, in association with a pharmaceutically acceptable diluent orcarrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof in combination with any oneof antihormonal agents listed under (ii) above, for example any one ofthe anti-oestrogens listed in (ii) above, or for example an aromataseinhibitor listed in (ii) above in association with a pharmaceuticallyacceptable diluent or carrier.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises Example 1 or a pharmaceutically acceptablesalt thereof and any one of antihormonal agents listed under (ii) above,for example any one of the anti-oestrogens listed in (ii) above, or forexample an aromatase inhibitor listed in (ii) above; in association witha pharmaceutically acceptable diluent or carrier.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising Example 1 or a pharmaceutically acceptable saltthereof and an mTOR inhibitor, such as AZD2014 (see for exampleWO2008/023161); in association with a pharmaceutically acceptablediluent or carrier.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising Example 1, or a pharmaceutically-acceptable saltthereof and a PI3Kα-inhibitor, such as those PI3K α/δ inhibitors in ourco-pending PCT application PCT/GB2014/050163, in association with apharmaceutically acceptable diluent or carrier. One example of asuitable PI3K α/δ inhibitor is Example 3 from PCT/GB2014/050163, asdescribed hereinbefore.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising Example 1 or a pharmaceutically acceptable saltthereof and palbociclib in association with a pharmaceuticallyacceptable diluent or carrier.

In one aspect the above pharmaceutical compositions of a compound offormula (I) or a pharmaceutically acceptable salt thereof, particularlyExample 1 or a pharmaceutically acceptable salt thereof, with ananti-tumour agent listed in (ii) above, or an mTOR inhibitor (such asAZD2014), or a PI3K-α inhibitor (such as those PI3K α/δ inhibitors inour co-pending PCT application PCT/GB2014/050163, particularly Example 3therein) or palbociclib, is suitable for use in the treatment of breastor gynaecological cancers, such as cancer of the breast, endometrium,ovary or cervix, particularly breast cancer, such as ER+ve breastcancer.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier foruse in treating cancer.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof in combination with any oneof antihormonal agents listed under (ii) above, for example any one ofthe anti-oestrogens listed in (ii) above, or for example an aromataseinhibitor listed in (ii) above in association with a pharmaceuticallyacceptable diluent or carrier for use in treating cancer.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises Example 1 or apharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier foruse in treating cancer.

In a further aspect of the invention there is provided a pharmaceuticalcomposition which comprises Example 1 or a pharmaceutically acceptablesalt thereof and any one of antihormonal agents listed under (ii) above,for example any one of the anti-oestrogens listed in (ii) above, or forexample an aromatase inhibitor listed in (ii) above; in association witha pharmaceutically acceptable diluent or carrier for use in treatingcancer.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising Example 1 or a pharmaceutically acceptable saltthereof and an mTOR inhibitor, such as AZD2014 (see for exampleWO2008/023161); in association with a pharmaceutically acceptablediluent or carrier for use in treating cancer.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising Example 1, or a pharmaceutically-acceptable saltthereof and a PI3Kα-inhibitor, such as those PI3K α/δ inhibitors in ourco-pending PCT application PCT/GB2014/050163, in association with apharmaceutically acceptable diluent or carrier for use in treatingcancer. One example of a suitable PI3K α/δ inhibitor is Example 3 fromPCT/GB2014/050163, as described hereinbefore.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising Example 1 or a pharmaceutically acceptable saltthereof and palbociclib in association with a pharmaceuticallyacceptable diluent or carrier for use in treating cancer.

In one aspect the above pharmaceutical compositions of a compound offormula (I) or a pharmaceutically acceptable salt thereof, particularlyExample 1 or a pharmaceutically acceptable salt thereof, with ananti-tumour agent listed in (ii) above, or an mTOR inhibitor (such asAZD2014), or a PI3K-α inhibitor (such as those PI3K α/δ inhibitors inour co-pending PCT application PCT/GB2014/050163, particularly Example 3therein) or palbociclib, is suitable for use in the treatment of breastor gynaecological cancers, such as cancer of the breast, endometrium,ovary or cervix, particularly breast cancer, such as ER+ve breastcancer.

According to another feature of the invention there is provided the useof a compound of the Formula (I) or a pharmaceutically acceptable saltthereof in combination with an anti-tumour agent selected from onelisted under (i)-(xi) herein above, in the manufacture of a medicamentfor use in the treatment of cancer in a warm-blooded animal, such asman.

According to another feature of the invention there is provided the useof Example 1 or a pharmaceutically acceptable salt thereof incombination with an anti-tumour agent selected from one listed under(i)-(xi) herein above, in the manufacture of a medicament for use incancer in a warm-blooded animal, such as man.

According to a further aspect of the invention there is provided the useof a compound of Formula (I) or a pharmaceutically acceptable saltthereof in combination with any one of antihormonal agents listed under(ii) above, for example any one of the anti-oestrogens listed in (ii)above, or for example an aromatase inhibitor listed in (ii) above in themanufacture of a medicament for use in the treatment of cancer in awarm-blooded animal, such as man.

In a further aspect of the invention there is provided the use ofExample 1 or a pharmaceutically acceptable salt thereof in combinationwith any one of antihormonal agents listed under (ii) above, for exampleany one of the anti-oestrogens listed in (ii) above, or for example anaromatase inhibitor listed in (ii) above; in the manufacture of amedicament for use in the treatment of cancer in a warm-blooded animal,such as man.

In a further aspect of the invention there is provided the use ofExample 1 or a pharmaceutically acceptable salt thereof in combinationwith an mTOR inhibitor, such as AZD2014 (see for example WO2008/023161);in the manufacture of a medicament for use in the treatment of cancer ina warm-blooded animal, such as man.

In a further aspect of the invention there is provided the use ofExample 1, or a pharmaceutically-acceptable salt thereof in combinationwith a PI3Kα-inhibitor, such as those PI3K α/δ inhibitors in ourco-pending PCT application PCT/GB2014/050163, in the manufacture of amedicament for use in the treatment of cancer in a warm-blooded animal,such as man. One example of a suitable PI3K α/δ inhibitor is Example 3from PCT/GB2014/050163, as described hereinbefore.

In a further aspect of the invention there is provided the use ofExample 1 or a pharmaceutically acceptable salt thereof in combinationwith palbociclib in the manufacture of a medicament for use in thetreatment of cancer in a warm-blooded animal, such as man.

In one aspect the above uses of a compound of formula (I) or apharmaceutically acceptable salt thereof, particularly Example 1 or apharmaceutically acceptable salt thereof, in combination with ananti-tumour agent listed in (ii) above, or an mTOR inhibitor (such asAZD2014), or a PI3K-α inhibitor (such as those PI3K α/δ inhibitors inour co-pending PCT application PCT/GB2014/050163, particularly Example 3therein) or palbociclib, is suitable for use in the manufacture of amedicament for the treatment of breast or gynaecological cancers, suchas cancer of the breast, endometrium, ovary or cervix, particularlybreast cancer, such as ER+ve breast cancer.

Therefore in an additional feature of the invention, there is provided amethod of treating cancer in a warm-blooded animal, such as man, in needof such treatment which comprises administering to said animal aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in combination with an anti-tumour agentselected from one listed under (i)-(xi) herein above.

According to a further aspect of the invention, there is provided amethod of treating cancer in a warm-blooded animal, such as man, in needof such treatment which comprises administering to said animal aneffective amount of Example 1 or a pharmaceutically acceptable saltthereof in combination with an anti-tumour agent selected from onelisted under (i)-(xi) herein above.

According to a further aspect of the invention there is provided amethod of treating cancer in a warm-blooded animal, such as man, in needof such treatment which comprises administering to said animal aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in combination with any one of antihormonalagents listed under (ii) above, for example any one of theanti-oestrogens listed in (ii) above, or for example an aromataseinhibitor listed in (ii) above.

In a further aspect of the invention there is provided a method oftreating cancer in a warm-blooded animal, such as man, in need of suchtreatment which comprises administering to said animal an effectiveamount of Example 1 or a pharmaceutically acceptable salt thereof incombination with any one of antihormonal agents listed under (ii) above,for example any one of the anti-oestrogens listed in (ii) above, or forexample an aromatase inhibitor listed in (ii) above.

In a further aspect of the invention there is provided a method oftreating cancer in a warm-blooded animal, such as man, in need of suchtreatment which comprises administering to said animal an effectiveamount of Example 1 or a pharmaceutically acceptable salt thereof incombination with an mTOR inhibitor, such as AZD2014 (see for exampleWO2008/023161).

In a further aspect of the invention there provided a method of treatingcancer in a warm-blooded animal, such as man, in need of such treatmentwhich comprises administering to said animal an effective amount ofExample 1, or a pharmaceutically-acceptable salt thereof in combinationwith a PI3Kα-inhibitor, such as those PI3K α/δ inhibitors in ourco-pending PCT application PCT/GB2014/050163. One example of a suitablePI3K α/δ inhibitor is Example 3 from PCT/GB2014/050163, as describedhereinbefore.

In a further aspect of the invention there is provided a method oftreating cancer in a warm-blooded animal, such as man, in need of suchtreatment which comprises administering to said animal an effectiveamount of Example 1 or a pharmaceutically acceptable salt thereof incombination with palbociclib.

In one aspect the above methods of treating cancer, are methods for thetreatment of breast or gynaecological cancers, such as cancer of thebreast, endometrium, ovary or cervix, particularly breast cancer, suchas ER+ve breast cancer.

According to a further aspect of the present invention there is provideda kit comprising a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in combination with an anti-tumour agentselected from one listed under (i)-(xi) herein above.

According to a further aspect of the present invention there is provideda kit comprising a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in combination with an anti-tumour agentselected from one listed under (i) or (ii) herein above.

According to a further aspect of the present invention there is provideda kit comprising:

-   a) a compound of Formula (I) or a pharmaceutically acceptable salt    thereof in a first unit dosage form;-   b) an anti-tumour agent selected from one listed under (i)-(xi)    herein above in a second unit dosage form; and-   c) container means for containing said first and second dosage    forms.

According to a further aspect of the present invention there is provideda kit comprising:

-   a) a compound of Formula (I) or a pharmaceutically acceptable salt    thereof in a first unit dosage form;-   b) an anti-tumour agent selected from one listed under (i)-(ii)    herein above in a second unit dosage form; and-   c) container means for containing said first and second dosage    forms.

According to a further aspect of the present invention there is provideda kit comprising Example 1 or a pharmaceutically acceptable salt thereofin combination with an anti-tumour agent selected from one listed under(i)-(xi) herein above.

According to a further aspect of the present invention there is provideda kit comprising Example 1 or a pharmaceutically acceptable salt thereofin combination with an anti-tumour agent selected from one listed under(i) or (ii) herein above.

According to a further aspect of the present invention there is provideda kit comprising:

-   a) Example 1 or a pharmaceutically acceptable salt thereof in a    first unit dosage form;-   b) an anti-tumour agent selected from one listed under (i)-(xi)    herein above in a second unit dosage form; and-   c) container means for containing said first and second dosage    forms.

According to a further aspect of the present invention there is provideda kit comprising:

-   a) Example 1 or a pharmaceutically acceptable salt thereof in a    first unit dosage form;-   b) an anti-tumour agent selected from one listed under (i)-(ii)    herein above in a second unit dosage form; and-   c) container means for containing said first and second dosage    forms.

According to a further aspect of the present invention there is provideda kit comprising:

-   a) Example 1 or a pharmaceutically acceptable salt thereof in a    first unit dosage form;-   b) an anti-tumour agent selected from AZD2014, a PI3Kα-inhibitor    (such as those PI3K α/δ inhibitors in our co-pending PCT application    PCT/GB2014/050163) and palbociclib in a second unit dosage form; and-   c) container means for containing said first and second dosage    forms.

In all of the above methods, uses and other aspects, where the compoundof Example 1 is used, it is suitably used as crystalline Form B.

Combination therapy as described above may be added on top of standardof care therapy typically carried out according to its usual prescribingschedule.

Although the compounds of the Formula (I) are primarily of value astherapeutic agents for use in warm-blooded animals (including man), theyare also useful whenever it is required to inhibit ER-α. Thus, they areuseful as pharmacological standards for use in the development of newbiological tests and in the search for new pharmacological agents.

Personalised Healthcare

Another aspect of the present invention is based on identifying a linkbetween the status of the gene encoding ERα and potential susceptibilityto treatment with a compound of Formula (I). In particular, ERα genestatus may indicate that a patient is less likely to respond to existinghormone therapy (such as aromatase inhibitors), in part at least becausesome ERα mutations are though to arise as resistance mechanisms toexisting treatments. A SERD, particularly a SERD which can beadministered orally in potentially larger doses without excessiveinconvenience, may then advantageously be used to treat patients withERα mutations who may be resistant to other therapies. This thereforeprovides opportunities, methods and tools for selecting patients fortreatment with a compound of Formula (I), particularly cancer patients.The present invention relates to patient selection tools and methods(including personalised medicine). The selection is based on whether thetumour cells to be treated possess wild-type or mutant ERα gene. The ERαgene status could therefore be used as a biomarker to indicate thatselecting treatment with a SERD may be advantageous. For the avoidanceof doubt, compounds of the formula (I) as described herein are thoughtto be similarly active against wild-type and mutant ERα genes, at leastthose mutations in ERα gene identified at the date of filing thisapplication.

There is a clear need for biomarkers that will enrich for or selectpatients whose tumours will respond to treatment with a SERD, such as acompound of Formula (I). Patient selection biomarkers that identify thepatients most likely to respond to one agent over another are ideal inthe treatment of cancer, since they reduce the unnecessary treatment ofpatients with non-responding tumours to the potential side effects ofsuch agents.

A biomarker can be described as “a characteristic that is objectivelymeasured and evaluated as an indicator of normal biologic processes,pathogenic processes, or pharmacologic responses to a therapeuticintervention”. A biomarker is any identifiable and measurable indicatorassociated with a particular condition or disease where there is acorrelation between the presence or level of the biomarker and someaspect of the condition or disease (including the presence of, the levelor changing level of, the type of, the stage of, the susceptibility tothe condition or disease, or the responsiveness to a drug used fortreating the condition or disease). The correlation may be qualitative,quantitative, or both qualitative and quantitative. Typically abiomarker is a compound, compound fragment or group of compounds. Suchcompounds may be any compounds found in or produced by an organism,including proteins (and peptides), nucleic acids and other compounds.

Biomarkers may have a predictive power, and as such may be used topredict or detect the presence, level, type or stage of particularconditions or diseases (including the presence or level of particularmicroorganisms or toxins), the susceptibility (including geneticsusceptibility) to particular conditions or diseases, or the response toparticular treatments (including drug treatments). It is thought thatbiomarkers will play an increasingly important role in the future ofdrug discovery and development, by improving the efficiency of researchand development programs. Biomarkers can be used as diagnostic agents,monitors of disease progression, monitors of treatment and predictors ofclinical outcome. For example, various biomarker research projects areattempting to identify markers of specific cancers and of specificcardiovascular and immunological diseases. It is believed that thedevelopment of new validated biomarkers will lead both to significantreductions in healthcare and drug development costs and to significantimprovements in treatment for a wide variety of diseases and conditions.

In order to optimally design clinical trials and to gain the mostinformation from these trials, a biomarker may be required. The markermay be measurable in surrogate and tumour tissues. Ideally these markerswill also correlate with efficacy and thus could ultimately be used forpatient selection.

Thus, the technical problem underlying this aspect of the presentinvention is the identification of means for stratification of patientsfor treatment with a compound of Formula (I). The technical problem issolved by provision of the embodiments characterized in the claimsand/or description herein.

Tumours which contain wild type ERα are believed to be susceptible totreatment with a compound of formula (I), for example as a first-linetreatment. Tumours may also respond to treatment with a compound offormula (I) as a second-line, third-line or subsequent therapy and thismay be useful, in particular, where the tumours contain mutant ERα andmay thus be resistant to existing therapies such as AIs. A higher dosageof a compound of formula (I) may be required in the resistant settingthan in wild type tumours).

The invention provides a method of determining sensitivity of cells to acompound of Formula (I). The method comprises determining the status ofERα gene in said cells. A cell is defined as sensitive to a compound ofFormula (I) if it inhibits the increase in cell number in a cell growthassay (either through inhibition of cell proliferation and/or throughincreased cell death). Methods of the invention are useful forpredicting which cells are more likely to respond to a compound ofFormula (I) by growth inhibition.

A sample “representative of the tumour” can be the actual tumour sampleisolated, or may be a sample that has been further processed, e.g. asample of PCR amplified nucleic acid from the tumour sample.

Definitions:

In this Personalised Healthcare section:

“Allele” refers to a particular form of a genetic locus, distinguishedfrom other forms by its particular nucleotide or amino acid sequence.

“Amplification reactions” are nucleic acid reactions which result inspecific amplification of target nucleic acids over non-target nucleicacids. The polymerase chain reaction (PCR) is a well known amplificationreaction.

“Cancer” is used herein to refer to neoplastic growth arising fromcellular transformation to a neoplastic phenotype. Such cellulartransformation often involves genetic mutation.

“Gene” is a segment of DNA that contains all the information for theregulated biosynthesis of an RNA product, including a promoter, exons,introns, and other sequence elements which may be located within 5′ or3′ flanking regions (not within the transcribed portions of the gene)that control expression.

“Gene status” refers to whether the gene is wild type or not (i.e.mutant).

“Label” refers to a composition capable of producing a detectable signalindicative of the presence of the target polynucleotide in an assaysample. Suitable labels include radioisotopes, nucleotide chromophores,enzymes, substrates, fluorescent molecules, chemiluminescent moieties,magnetic particles, bioluminescent moieties, and the like. As such, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.

“Non-synonymous variation” refers to a variation (variance) in oroverlapping the coding sequence of a gene that result in the productionof a distinct (altered) polypeptide sequence. These variations may ormay not affect protein function and include missense variants (resultingin substitution of one amino acid for another), nonsense variants(resulting in a truncated polypeptide due to generation of a prematurestop codon) and insertion/deletion variants.

“Synonymous variation” refers to a variation (variance) in the codingsequence of a gene that does not affect sequence of the encodedpolypeptide. These variations may affect protein function indirectly(for example by altering expression of the gene), but, in the absence ofevidence to the contrary, are generally assumed to be innocuous.

“Nucleic acid” refers to single stranded or double stranded DNA and RNAmolecules including natural nucleic acids found in nature and/ormodified, artificial nucleic acids having modified backbones or bases,as are known in the art.

“Primer” refers to a single stranded DNA oligonucleotide sequencecapable of acting as a point of initiation for synthesis of a primerextension product which is complementary to the nucleic acid strand tobe copied. The length and sequence of the primer must be such that theyare able to prime the synthesis of extension products. A typical primercontains at least about 7 nucleotides in length of a sequencesubstantially complementary to the target sequence, but somewhat longerprimers are preferred. Usually primers contain about 15-26 nucleotides,but longer or shorter primers may also be employed.

“Polymorphic site” is a position within a locus at which at least twoalternative sequences are found in a population.

“Polymorphism” refers to the sequence variation observed in anindividual at a polymorphic site. Polymorphisms include nucleotidesubstitutions, insertions, deletions and microsatellites and may, butneed not, result in detectable differences in gene expression or proteinfunction. In the absence of evidence of an effect on expression orprotein function, common polymorphisms, including non-synonomousvariants, are generally considered to be included in the definition ofwild-type gene sequence. A catalog of human polymorphisms and associatedannotation, including validation, observed frequencies, and diseaseassociation, is maintained by NCBI (dbSNP:http://www.ncbi.nlm.nih.gov/projects/SNP/). Please note that the term“polymorphism” when used in the context of gene sequences should not beconfused with the term “polymorphism” when used in the context of solidstate form of a compound, that is the crystalline or amorphous nature ofa compound. The skilled person will understand the intended meaning byits context.

“Probe” refers to single stranded sequence-specific oligonucleotideswhich have a sequence that is exactly complementary to the targetsequence of the allele to be detected.

“Response” is defined by measurements taken according to ResponseEvaluation Criteria in Solid Tumours (RECIST) involving theclassification of patients into two main groups: those that show apartial response or stable disease and those that show signs ofprogressive disease.

“Stringent hybridisation conditions” refers to an overnight incubationat 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulphate, and 20 pg/mI denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

“Survival” encompasses a patients' overall survival and progression-freesurvival.

“Overall survival” (OS) is defined as the time from the initiation ofdrug administration to death from any cause. “Progression-free survival”(PFS) is defined as the time from the initiation of drug administrationto first appearance of progressive disease or death from any cause.

According to one aspect of the invention there is provided a method forselecting a patient for treatment with a compound of Formula (I), themethod comprising providing a tumour cell containing sample from apatient; determining whether the ERα gene in the patient's tumour cellcontaining sample is wild type or mutant; and selecting a patient fortreatment with a compound of Formula (I) based thereon.

The method may include or exclude the actual patient sample isolationstep. Thus, according to one aspect of the invention there is provided amethod for selecting a patient for treatment with a compound of Formula(I), the method comprising determining whether the ERα gene in a tumourcell containing sample previously isolated from the patient is wild typeor mutant; and selecting a patient for treatment with a compound ofFormula (I) based thereon.

In one embodiment, the patient is selected for treatment with a compoundof Formula (I) if the tumour cell DNA has a mutant ERα gene. In otherembodiments, a patient whose tumour cell DNA possesses a wild type ERαgene is selected for treatment with a compound of Formula (I).

For the purpose of this invention, a gene status of wild-type is meantto indicate normal or appropriate expression of the gene and normalfunction of the encoded protein.

In contrast, mutant status is meant to indicate expression of a proteinwith altered function, consistent with the known roles of mutant ERαgenes in cancer (as described herein). Any number of genetic orepigenetic alterations, including but not limited to mutation,amplification, deletion, genomic rearrangement, or changes inmethylation profile, may result in a mutant status. However, if suchalterations nevertheless result in appropriate expression of the normalprotein, or a functionally equivalent variant, then the gene status isregarded as wild-type. Examples of variants that typically would notresult in a functional mutant gene status include synonomous codingvariants and common polymorphisms (synonymous or non-synonymous). Asdiscussed below, gene status can be assessed by a functional assay, orit may be inferred from the nature of detected deviations from areference sequence.

In certain embodiments the wild-type or mutant status of the ERα gene isdetermined by the presence or absence of non-synonymous nucleic acidvariations in the genes. Observed non-synonymous variationscorresponding to known common polymorphisms with no annotated functionaleffects do not contribute to a gene status of mutant.

Other variations in the ERα gene that signify mutant status includesplice site variations that decrease recognition of an intron/exonjunction during processing of pre-mRNA to mRNA. This can result in exonskipping or the inclusion of normally intronic sequence in spliced mRNA(intron retention or utilization of cryptic splice junctions). This can,in turn, result in the production of aberrant protein with insertionsand/or deletions relative to the normal protein. Thus, in otherembodiments, the gene has a mutant status if there is a variant thatalters splice site recognition sequence at an intron/exon junction.

For ESR1, reference sequences are available for the gene (GenBankaccession number: NG_008493), mRNA (GenBank accession number:NM_000125), and protein (GenBank accession number: NP_000116 orSwiss-Prot accession: P03372). A person of skill in the art will be ableto determine the ESR1 gene status, i.e. whether a particular ESR1 geneis wild type or mutant, based on comparison of DNA or protein sequencewith wild type.

It will be apparent that the gene and mRNA sequences disclosed for ERαgene are representative sequences. In normal individuals there are twocopies of each gene, a maternal and paternal copy, which will likelyhave some sequence differences, moreover within a population there willexist numerous allelic variants of the gene sequence. Other sequencesregarded as wild type include those that possess one or more synonymouschanges to the nucleic acid sequence (which changes do not alter theencoded protein sequence), non-synonymous common polymorphisms (e.g.germ-line polymorphisms) which alter the protein sequence but do notaffect protein function, and intronic non-splice-site sequence changes.

There are numerous techniques available to the person skilled in the artto determine the gene status of ERα. The gene status can be determinedby determination of the nucleic acid sequence. This could be via directsequencing of the full-length gene or analysis of specific sites withinthe gene, e.g. commonly mutated sites.

Samples

The patient's sample to be tested for the gene status can be any tumourtissue or tumour-cell containing sample obtained or obtainable from theindividual. The test sample is conveniently a sample of blood, mouthswab, biopsy, or other body fluid or tissue obtained from an individual.Particular examples include: circulating tumour cells, circulating DNAin the plasma or serum, cells isolated from the ascites fluid of ovariancancer patients, lung sputum for patients with tumours within the lung,a fine needle aspirate from a breast cancer patient, urine, peripheralblood, a cell scraping, a hair follicle, a skin punch or a buccalsample.

It will be appreciated that the test sample may equally be a nucleicacid sequence corresponding to the sequence in the test sample, that isto say that all or a part of the region in the sample nucleic acid mayfirstly be amplified using any convenient technique e.g. polymerasechain reaction (PCR), before analysis. The nucleic acid may be genomicDNA or fractionated or whole cell RNA. In particular embodiments the RNAis whole cell RNA and is used directly as the template for labelling afirst strand cDNA using random primers or poly A primers. The nucleicacid or protein in the test sample may be extracted from the sampleaccording to standard methodologies (see Green & Sambrook, Eds.,Molecular Cloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3,ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

The diagnostic methods of the invention can be undertaken using a samplepreviously taken from the individual or patient. Such samples may bepreserved by freezing or fixed and embedded in formalin-paraffin orother media. Alternatively, a fresh tumour cell containing sample may beobtained and used.

The methods of the invention can be applied using cells from any tumour.Suitable tumours for treatment with a compound of Formula (I) have beendescribed hereinbefore.

Methods for Detection of Nucleic Acids

The detection of mutant ERα nucleic acids can be employed, in thecontext of the present invention, to select drug treatment. Sincemutations in these genes occur at the DNA level, the methods of theinvention can be based on detection of mutations or variances in genomicDNA, as well as transcripts and proteins themselves. It can be desirableto confirm mutations in genomic DNA by analysis of transcripts and/orpolypeptides, in order to ensure that the detected mutation is indeedexpressed in the subject.

It will be apparent to the person skilled in the art that there are alarge number of analytical procedures which may be used to detect thepresence or absence of variant nucleotides at one or more positions in agene. In general, the detection of allelic variation requires a mutationdiscrimination technique, optionally an amplification reaction (such asone based on polymerase chain reaction) and optionally a signalgeneration system. There are a multitude of mutation detectiontechniques available in the art and these may be used in combinationwith a signal generation system, of which there are numerous availablein the art. Many methods for the detection of allelic variation arereviewed by Nollau et al., Clin. Chem., 1997, 43, 1114-1120; AndersonSM. Expert Rev Mol Diagn., 2011, 11, 635-642; Meyerson M. et al., NatRev Genet., 2010, 11, 685-696; and in standard textbooks, for example“Laboratory Protocols for Mutation Detection”, Ed. by U. Landegren,Oxford University Press, 1996 and “PCR”, 2^(nd) Edition by Newton &Graham, BIOS Scientific Publishers Limited, 1997.

As noted above, determining the presence or absence of a particularvariance or plurality of variances in the ERα gene in a patient withcancer can be performed in a variety of ways. Such tests are commonlyperformed using DNA or RNA collected from biological samples, e.g.,tissue biopsies, urine, stool, sputum, blood, cells, tissue scrapings,breast aspirates or other cellular materials, and can be performed by avariety of methods including, but not limited to, PCR, hybridizationwith allele-specific probes, enzymatic mutation detection, chemicalcleavage of mismatches, mass spectrometry or DNA sequencing, includingminisequencing.

Suitable mutation detection techniques include amplification refractorymutation system (ARMS™), amplification refractory mutation system linearextension (ALEX™), competitive oligonucleotide priming system (COPS),Taqman, Molecular Beacons, restriction fragment length polymorphism(RFLP), and restriction site based PCR and fluorescence resonance energytransfer (FRET) techniques.

In particular embodiments the method employed for determining thenucleotide(s) within a biomarker gene is selected from: allele-specificamplification (allele specific PCR)—such as amplification refractorymutation system (ARMS), sequencing, allelic discrimination assay,hybridisation, restriction fragment length polymorphism (RFLP) oroligonucleotide ligation assay (OLA).

In particular embodiments, hybridization with allele specific probes canbe conducted by: (1) allele specific oligonucleotides bound to a solidphase (e.g. glass, silicon, nylon membranes) with the labelled sample insolution, for example as in many DNA chip applications; or, (2) boundsample (often cloned DNA or PCR amplified DNA) and labelledoligonucleotides in solution (either allele specific or short so as toallow sequencing by hybridization). Diagnostic tests may involve a panelof variances, often on a solid support, which enables the simultaneousdetermination of more than one variance. Such hybridization probes arewell known in the art (see, e.g., Green & Sambrook, Eds., MolecularCloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN9781936113422), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) and may span two or more variance sites.

Thus, in one embodiment, the detection of the presence or absence of atleast one mutation provides for contacting ERα nucleic acid containing aputative mutation site with at least one nucleic acid probe. The probepreferentially hybridizes with a nucleic acid sequence including avariance site and containing complementary nucleotide bases at thevariance site under selective hybridization conditions. Hybridizationcan be detected with a detectable label using labels known to oneskilled in the art. Such labels include, but are not limited toradioactive, fluorescent, dye, and enzymatic labels.

In another embodiment, the detection of the presence or absence of atleast one mutation provides for contacting ERα nucleic acid containing aputative mutation site with at least one nucleic acid primer. The primerpreferentially hybridizes with a nucleic acid sequence including avariance site and containing complementary nucleotide bases at thevariance site under selective hybridization conditions.

Oligonucleotides used as primers for specific amplification may carrythe complementary nucleotide base to the mutation of interest in thecentre of the molecule (so that amplification depends on differentialhybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res., 17,2437-248) or at the extreme 3′-terminus of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (see, e.g., Prossner, 1993, Tibtech, 11 238).

In yet another embodiment, the detection of the presence or absence ofat least one mutation comprises sequencing at least one nucleic acidsequence and comparing the obtained sequence with the known wild typenucleic acid sequence.

Alternatively, the presence or absence of at least one mutationcomprises mass spectrometric determination of at least one nucleic acidsequence.

In one embodiment, the detection of the presence or absence of at leastone nucleic acid variance comprises performing a polymerase chainreaction (PCR). The target nucleic acid sequence containing thehypothetical variance is amplified and the nucleotide sequence of theamplified nucleic acid is determined. Determining the nucleotidesequence of the amplified nucleic acid comprises sequencing at least onenucleic acid segment. Alternatively, amplification products can beanalyzed using any method capable of separating the amplificationproducts according to their size, including automated and manual gelelectrophoresis, and the like.

Mutations in genomic nucleic acid are advantageously detected bytechniques based on mobility shift in amplified nucleic acid fragments.For instance, Chen et al., Anal Biochem 1996, 239, 61-9, describe thedetection of single-base mutations by a competitive mobility shiftassay. Moreover, assays based on the technique of Marcelino et al.,BioTechniques 1999, 26, 1134-1148 are available commercially.

In a particular example, capillary heteroduplex analysis may be used todetect the presence of mutations based on mobility shift of duplexnucleic acids in capillary systems as a result of the presence ofmismatches.

Generation of nucleic acids for analysis from samples generally requiresnucleic acid amplification. Many amplification methods rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.Preferably, the amplification according to the invention is anexponential amplification, as exhibited by for example the polymerasechain reaction.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U., et al., Science, 1988 242, 229-237 and Lewis, R., GeneticEngineering News 1990, 10, 54-55. These amplification methods can beused in the methods of our invention, and include polymerase chainreaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligasehybridisation, Qβ bacteriophage replicase, transcription-basedamplification system (TAS), genomic amplification with transcriptsequencing (GAWTS), nucleic acid sequence-based amplification (NASBA)and in situ hybridisation. Primers suitable for use in variousamplification techniques can be prepared according to methods known inthe art.

Polymerase Chain Reaction (PCR) PCR is a nucleic acid amplificationmethod described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.PCR consists of repeated cycles of DNA polymerase generated primerextension reactions. The target DNA is heat denatured and twooligonucleotides, which bracket the target sequence on opposite strandsof the DNA to be amplified, are hybridised. These oligonucleotidesbecome primers for use with DNA polymerase. The DNA is copied by primerextension to make a second copy of both strands. By repeating the cycleof heat denaturation, primer hybridisation and extension, the target DNAcan be amplified a million fold or more in about two to four hours. PCRis a molecular biology tool, which must be used in conjunction with adetection technique to determine the results of amplification. Anadvantage of PCR is that it increases sensitivity by amplifying theamount of target DNA by 1 million to 1 billion fold in approximately 4hours. PCR can be used to amplify any known nucleic acid in a diagnosticcontext (Mok et al., Gynaecologic Oncology, 1994, 52: 247-252,).

An allele specific amplification technique such as AmplificationRefractory Mutation System (ARMS™) (Newton et al., Nucleic Acids Res.,1989, 17, 2503-2516) can also be used to detect single base mutations.Under the appropriate PCR amplification conditions a single basemismatch located at the 3′-end of the primer is sufficient forpreferential amplification of the perfectly matched allele (Newton etal., 1989, supra), allowing the discrimination of closely relatedspecies. The basis of an amplification system using the primersdescribed above is that oligonucleotides with a mismatched 3′-residuewill not function as primers in the PCR under appropriate conditions.This amplification system allows genotyping solely by inspection ofreaction mixtures after agarose gel electrophoresis.

Analysis of amplification products can be performed using any methodcapable of separating the amplification products according to theirsize, including automated and manual gel electrophoresis, massspectrometry, and the like.

The methods of nucleic acid isolation, amplification and analysis areroutine for one skilled in the art and examples of protocols can befound, for example, Green & Sambrook, Eds., Molecular Cloning: ALaboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN 9781936113422),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)Particularly useful protocol source for methods used in PCRamplification is PCR (Basics: From Background to Bench) by M. J.McPherson, S. G. Mailer, R. Beynon, C. Howe, Springer Verlag; 1stedition (Oct. 15, 2000), ISBN: 0387916008.

The present invention also provides predictive and diagnostic kitscomprising degenerate primers to amplify a target nucleic acid in theERα gene and instructions comprising; amplification protocol andanalysis of the results. The kit may alternatively also comprisebuffers, enzymes, and containers for performing the amplification andanalysis of the amplification products. The kit may also be a componentof a screening, or diagnostic kit comprising other tools such as DNAmicroarrays, or other supports. Preferably, the kit also provides one ormore control templates, such as nucleic acids isolated from normaltissue sample, and/or a series of samples representing differentvariances in the reference genes.

In one embodiment, the kit provides two or more primer pairs, each paircapable of amplifying a different region of the reference (ERα) gene(each region a site of potential variance) thereby providing a kit foranalysis of expression of several gene variances in a biological samplein one reaction or several parallel reactions.

Primers in the kits may be labelled, for example fluorescently labelled,to facilitate detection of the amplification products and consequentanalysis of the nucleic acid variances. The kit may also allow for morethan one variance to be detected in one analysis. A combination kit willtherefore comprise of primers capable of amplifying different segmentsof the reference gene. The primers may be differentially labelled, forexample using different fluorescent labels, so as to differentiatebetween the variances.

In another aspect, the invention provides a method of treating a patientsuffering from cancer comprising: determining the mutant or wild typestatus of the ERα gene in the patient's tumour cells and if the ERα geneis mutant, administering to the patient an effective amount of acompound of Formula (I).

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. “Lesseffective” means that the treatment results in a therapeuticallysignificant lower level of pharmacological effectiveness and/or atherapeutically greater level of adverse physiological effects.

According to another aspect of the invention there is provided the useof a compound of Formula (I) or a pharmaceutically-acceptable saltthereof to treat a cancer patient whose tumour cells have beenidentified as possessing a mutant ERα gene. In one embodiment thecompound of Formula (I) is Example 1.

According to another aspect of the invention there is provided acompound of Formula (I) or a pharmaceutically-acceptable salt thereoffor treating cancers with tumour cells identified as harbouring mutantERα gene. In one embodiment the compound of Formula (I) is Example 1.

According to another aspect of the invention there is provided a methodof treating cancers with tumour cells identified as harbouring mutantERα gene comprising administering an effective amount of a compound ofFormula (I) or a pharmaceutically-acceptable salt thereof. In oneembodiment the compound of Formula (I) is Example 1.

In still further embodiments, the invention relates to pharmaceuticalcomposition comprising a compound of Formula (I) for use in theprevention and treatment of cancer with tumour cells identified asharbouring a mutant ERα gene. In one embodiment the compound of Formula(I) is Example 1.

For all the aspects above, mutant forms of ERα determined/identified areat all positions across the gene.

For all the aspects above, using tumours such as breast cancer as anexample, particular mutant forms of ERα determined/identified are thoseat positions Ser463Pro, Val543Glu, Leu536Arg, Tyr537Ser, Tyr537Asn andAsp538Gly.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an X-Ray Powder Diffraction Pattern of Example 7 Form A

FIG. 2 shows an X-Ray Powder Diffraction Pattern of Example 1 Form B

FIG. 3 shows DSC Thermogram of Example 1 Form B

FIG. 4 shows X-Ray Powder Diffraction Pattern of Example 1 Form A

FIG. 5 shows TGA Thermogram of Example 1 Form A

FIG. 6 shows X-Ray Powder Diffraction Pattern of Example 1 Form C

FIG. 7 shows TGA Thermogram of Example 1 Form C

FIG. 8 shows an X-Ray Powder Diffraction Pattern of Example 11

FIG. 9 shows a DSC trace of Example 11.

FIG. 10 shows the results of an MCF-7 xenograft study with Example 1 andAZD2014.

FIGS. 11 and 12 show the results of an HCC1428 long term estrogendeprived (LTED) xenograft efficacy study with Example 1.

EXAMPLES

The invention will now be illustrated in the following Examples inwhich, generally:

-   -   (i) operations were carried out at ambient temperature, i.e. in        the range 17 to 25° C. and under an atmosphere of an inert gas        such as nitrogen unless otherwise stated;    -   (ii) evaporations were carried out by rotary evaporation or        utilising Genevac equipment or Biotage v10 evaporator in vacuo        and work-up procedures were carried out after removal of        residual solids by filtration;    -   (iii) flash chromatography purifications were performed on an        automated Teledyne Isco CombiFlash® Rf or Teledyne Isco        CombiFlash® Companion® using prepacked RediSep Rf Gold™ Silica        Columns (20-40 m, spherical particles), GraceResolv™ Cartridges        (Davisil® silica) or Silicycle cartridges (40-63 m).    -   (iv) preparative chromatography was performed on a Gilson prep        HPLC instrument with UV collection;    -   (v) chiral preparative chromatography was performed on a Gilson        instrument with UV collection (233 injector/fraction collector,        333 & 334 pumps, 155 UV detector) or a Varian Prep Star        instrument (2×SD1 pumps, 325 UV detector, 701 fraction        collector) pump running with Gilson 305 injection;    -   (vi) yields, where present, are not necessarily the maximum        attainable;    -   (vii) in general, the structures of end-products of the Formula        I were confirmed by nuclear magnetic resonance (NMR)        spectroscopy; NMR chemical shift values were measured on the        delta scale [proton magnetic resonance spectra were determined        using a Bruker Avance 500 (500 MHz) or Bruker Avance 400 (400        MHz) instrument]; measurements were taken at ambient temperature        unless otherwise specified; the following abbreviations have        been used: s, singlet; d, doublet; t, triplet; q, quartet; m,        multiplet; dd, doublet of doublets; ddd, doublet of doublet of        doublet; dt, doublet of triplets; bs, broad signal    -   (viii) in general, end-products of the Formula I were also        characterised by mass spectroscopy following liquid        chromatography (LCMS or UPLC); UPLC was carried out using a        Waters UPLC fitted with Waters SQ mass spectrometer (Column temp        40, UV=220-300 nm, Mass Spec=ESI with positive/negative        switching) at a flow rate of 1 ml/min using a solvent system of        97% A+3% B to 3% A to 97% B over 1.50 mins (total runtime with        equilibration back to starting conditions etc 1.70 min), where        A=0.1% formic acid in water (for acid work) or 0.1% ammonia in        water (for base work) B=acetonitrile. For acid analysis the        column used was Waters Acquity HSS T3 1.8 μm 2.1×50 mm, for base        analysis the column used was Waters Acquity BEH 1.7 m 2.1×50 mm;        LCMS was carried out using a Waters Alliance HT (2795) fitted        with a Waters ZQ ESCi mass spectrometer and a Phenomenex        Gemini-NX (50×2.1 mm 5 μm) column at a flow rate of 1.1 ml/min        95% A to 95% B over 4 min with a 0.5 min hold. The modifier is        kept at a constant 5% C (50:50 acetonitrile:water 0.1% formic        acid) or D (50:50 acetonitrile:water 0.1% ammonium hydroxide        (0.88 SG) depending on whether it is an acidic or basic method.    -   (ix) ion exchange purification was generally performed using a        SCX-2 (Biotage, Propylsulfonic acid functionalized silica.        Manufactured using a trifunctional silane. Non end-capped)        cartridge.    -   (x) intermediates purity was assessed by thin layer        chromatographic, mass spectral, HPLC (high performance liquid        chromatography) and/or NMR analysis;    -   (xi) For XRPD analysis of Example 7, samples were mounted on        zero background silicon wafers and analysed using the        PANalytical CubiX Pro diffractometer (1=1.5418 Å). Samples were        spun to improve counting statistics. Data was collected in        reflection geometry in theta-2theta configuration over the scan        range 2° to 40° 2-theta with 25 second exposure per 0.025067°        increment. X-rays were generated by a copper long-fine focus        tube operated at 45 kV and 40 mA. Persons skilled in the art of        X-ray powder diffraction will realise that the relative        intensity of peaks can be affected by, for example, grains above        30 microns in size and non-unitary aspect ratios that may affect        analysis of samples. The skilled person will also realise that        the position of reflections can be affected by the precise        height at which the sample sits in the diffractometer and the        zero calibration of the diffractometer. The surface planarity of        the sample may also have a small effect. Hence the diffraction        pattern data presented are not to be taken as absolute values;    -   (xii) For XRPD analysis of Example 1, The X-ray powder        diffractogram was determined by mounting a sample of the        crystalline material on a Panalytical single silicon crystal        (SSC) wafer mount and spreading out the sample into a thin layer        with the aid of a microscope slide. The sample was spun at 30        revolutions per minute (to improve counting statistics) and        irradiated with X-rays generated by a copper long-fine focus        tube operated at 45 kV and 40 mA with a wavelength of 1.5418        angstroms. The X-ray beam was passed through a 0.04 rad soller        slit, then an automatic variable divergence slit set at 20 mm        and finally a 20 mm beam mask. The reflected radiation was        directed through a 20 mm antiscatter slit and a 0.04 rad soller        slit. The sample was exposed for 1.905 seconds per 0.0025067°        2-theta increment (continuous scan mode) over the range 2        degrees to 40 degrees 2-theta in theta-theta mode. The running        time was 3 minutes and 36 seconds. The instrument was equipped        X-Celerator detector. Control and data capture was by means of a        Dell Pentium 4HT Workstation operating with X'Pert Industry        software.    -   (xiii) Differential Scanning Calorimetry: Analytical Instrument:        TA Instruments Q1000 DSC. Typically less than 5 mg of material        contained in a standard aluminium pan fitted with a lid was        heated over the temperature range 25° C. to 300° C. at a        constant heating rate of 10° C. per minute. A purge gas using        nitrogen was used-flow rate 50 ml per minute.    -   (xiv) Thermogravimetric Analysis Analytical Instrument: TA        Instruments Q5000 TGA. Typically less than 10 mg of material is        placed on a 100 μl platinum pan and heated over the temperature        range 30° C. to 150° C. at a constant heating rate of 10° C. per        minute.    -   (xv) the following abbreviations have been used:—        -   aq. aqueous        -   CDCl₃ deutero-chloroform        -   Conc. concentrated        -   DCM dichloromethane        -   DMA N,N-dimethylacetamide        -   DMSO dimethyl sulphoxide        -   DSC differential scanning calorimetry,        -   EtOH ethanol        -   EtOAc ethyl acetate        -   IPA/iPrOH isopropyl alcohol        -   MeCN acetonitrile        -   MTBE methyltertbutyl ether        -   rt/RT room temperature        -   sat. saturated        -   sol. solution        -   THF tetrahydrofuran        -   TFA trifluoroacetic acid        -   TGA Thermogravimetric analysis

Example 1(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

The following processes should be carried out under an atmosphere ofnitrogen in the absence of light as a light degradation product[(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate]may be formed.

(E)-Methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(350 g, 766.69 mmol) was charged to a 5 L fixed vessel. Isopropylalcohol (2.80 L) was added to the vessel. Sodium hydroxide (5M, 460 ml,2.30 mol) was added in one portion and the mixture was stirred at 21° C.for 16 hrs. The dark solution was screened through a filter to removeparticulates. The filtrate was returned to the reactor vessel. Thefilter and filtrate collection vessel were washed with isopropanol (700ml) and the washings were added to the reactor vessel. The reactionmixture was agitated and water (1.75 L) was added. Concentratedhydrochloric acid (37% w/w, 165 ml, 1.92 mol) was charged to the vessel.Further hydrochloric acid (21.5 ml) was added to the vessel to adjustthe pH between 4.0 and 4.5. The solution was heated to 50° C. Water(1.92 L) added to the vessel over 1 hour maintaining the internaltemperature between 50-53° C. The jacket temperature was raised to 70°C. to maintain the reactor temperature in this range during theaddition. Within 10 minutes of completion of the water addition, themixture self seeded and started to crystallise. The mixture was held at50-52° C. for 1.5 hours (jacket set temperature 58° C.). The resultingyellow suspension was cooled to 5° C. (jacket temperature) over 6 hours.The slurry was held at 5° C. (jacket temperature) for 11 hrs. Theresultant yellow solid was isolated by filtration. The cake was pastedwith a spatula to prevent cracking of the cake. The vessel was washedwith water (1.05 L). The washings were used to wash the cake. The cakewas pulled dry in air then dried in vacuo to constant weight over 4 days(oven temperature=30° C.).(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid was thus isolated as a yellow crystalline solid, “Form B” (319.45g, 94%). ¹H NMR (500 MHz, DMSO, 27° C.) 1.05 (3H, d), 1.08-1.28 (6H, m),2.35 (1H, dd), 2.58 (1H, dd), 2.8-2.97 (2H, m), 3.47-3.57 (1H, m), 5.22(1H, s), 6.67 (1H, d), 6.91-7.06 (2H, m), 7.19 (1H, d), 7.41 (1H, d),7.46 (2H, d), 7.54 (1H, d), 10.58 (1H, s), 12.62 (1H, s).

An alternative method for synthesising Example 1, which results information of Form B crystalline material, is as follows:

The following processes should be carried out under an atmosphere ofnitrogen in the absence of light as a light degradation product (asdescribed above) may be formed. (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(50.0 g; 109.53 mmol) was stirred in isopropyl alcohol (450 ml). Sodiumhydroxide (68.34 g, 65.72 ml, 328.58 mmol) was added in one portion andthe mixture stirred at 20° C. for 16 hrs. The reaction mixture wasdiluted with water (250 ml), the pH adjusted to pH4 with conc.hydrochloric acid (27.28 ml, 317.63 mmol) and the mixture heated to 50°C. Further water (225 ml) was added over 30 minutes, maintaining thetemperature above 45° C. During the addition, the material began tocrystallise. The mixture was cooled from 50° C. to 5° C. over 5 hoursthen the suspension was held at 0° C. for a further 11 hours. The yellowsolid was isolated by filtration. The filter cake was washed with water(100 ml), dried on the filter for a further 20 minutes then dried in avacuum oven for 16 hours to constant weight (30° C., air bleed) to givethe title compound (46.52 g) as a crystalline solid (Form B).

¹H NMR (500 MHz, DMSO, 27° C.) 1.02-1.09 (3H, m), 1.17 (6H, dd), 2.37(1H, dd), 2.59 (1H, dd), 2.8-2.98 (2H, m), 3.47-3.58 (1H, m), 5.24 (1H,s), 6.68 (1H, d), 6.9-7.06 (2H, m), 7.20 (1H, d), 7.38-7.51 (3H, m),7.55 (1H, d), 10.59 (1H, s), 12.60 (1H, br).

Crystalline form B may also be isolated from ethanol/water mixtures andethanol/MTBE mixtures.

In a further aspect of the invention there is provided crystalline formB of Example 1, isolated from isopropanol/water mixture.

In a further aspect of the invention, there is provided a process forisolation of crystalline form B of Example 1 which comprises hydrolysisof (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylatein isopropyl alcohol and base, followed by acidification and isolationof crystalline product from aqueous isopropanol.

Example 1 Form B is characterised in providing at least one of thefollowing 20 values measured using CuKα radiation: 8.4 and 10.9. Example1 Form B is characterised in providing an X-ray powder diffractionpattern, substantially as shown in FIG. 2. Ten X-Ray powder diffractionpeaks are shown in Table A:

TABLE A Ten X-Ray Powder Diffraction peaks for Example 1 Form B Angle 2-Theta (2θ) Intensity % 8.4 100 10.9 88.5 18.3 84.5 24.0 78.5 14.0 66.419.0 55.9 14.4 54.3 13.0 45 15.3 44.7 20.6 44.2

DSC analysis of Example 1 Form B shows it to be a high melting solidwith an endotherm showing onset of melting at 188.6° C. (FIG. 3).Example 1 shows degradation through the melt which may lead to variationin melting onset, thus the value of 188.6° C. should not be taken asabsolute.

Two solvated forms of Example 1 have also been observed.

Form A is a methyl tertiary butyl ether mono-solvate. The X-ray powderdiffraction pattern is shown in FIG. 4. TGA shows an associated weightloss of 14.7% w/w between 55-150° C. (FIG. 5). A theoretical loss for amono methyl tertiary butyl ether solvate is calculated to be 16.6%.

Form C is an Acetone mono-solvate, the X-ray powder diffraction patternis shown in FIG. 6. TGA shows an associated weight loss of 10.0% w/w50-150° C. %. (FIG. 7). The theoretical loss for a mono-acetone solvateis calculated to be 11.0%.

The (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateused as starting material was prepared as follows:—

Preparation of 2-fluoro-2-methylpropyl trifluoromethanesulfonate

Under an atmosphere of nitrogen, 2-fluoro-2-methyl-propan-1-ol (1000 g,10.86 moles) was charged to a 20 L vessel. DCM (8.5 L) was added to thevessel. The mixture was agitated and cooled to 1° C. 2,6-Lutidine (1395g, 13.02 moles) was added to the mixture. A solution oftrifluoromethanesulfonic anhydride (3220 g; 11.41 moles) in DCM (1 L)was added over 1 hour maintaining the temperature of the reactionmixture below 5° C. (the jacket set temperature was lowered to −20° C.during the addition). The addition vessel was and lines were washed withDCM (0.5 L) and the washings were added to the vessel in one portion.The mixture was agitated at 0° C. for 1 hour affording a red solution.

A solution of concentrated hydrochloric acid (1.23 L, 37% w/w, 16.3moles) was added to water (7 L). The dilute hydrochloric acid solutionwas added to the red solution and the stirred mixture was warmed to 25°C. The layers were allowed to separate and the upper aqueous layer wasdiscarded. The organic layer was washed with water (2×5 L). The organicsolution was concentrated under reduced pressure to afford a red oil.The red oil was purified by distillation using a wiped film evaporator(4.5 mbar, jacket temperature 50° C., condenser temperature 4° C.) toafford 2-fluoro-2-methylpropyl trifluoromethanesulfonate (1.69 Kg, 69%)as a pale red oil. ¹H NMR (500 MHz, DMSO-d6, 27° C.) δ 1.40 (6H, d),4.79 (2H, d).

Preparation of(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine

(2R)-1-(1H-Indol-3-yl)propan-2-amine (3.81 kg, 21.21 moles) was added toa 100 L glass lined jacketed vessel under an atmosphere of nitrogen.1,4-dioxane (23 L) was added, and the agitator was switched on.Diisopropylethylamine (5.55 L; 31.82 moles) was added to the stirredsuspension followed by(2-fluoro-2-methyl-propyl)trifluoromethanesulfonate (5.55 kg, 23.77moles). 1,4-Dioxane (4 L) was added to the vessel, and the mixture washeated to 75° C. Heating was continued for 24 hours before cooling themixture to 25° C. Water (30.5 L) was added to the vessel, followed bytoluene (30.5 L). After 40 minutes the agitator was switched off and thelayers were allowed to separate. The aqueous layer was removed and water(30.5 L) was added to the organic solution. The mixture was agitated for15 minutes before allowing the layers to separate. The aqueous layer wasremoved from the vessel. The organic solution was concentrated by vacuumdistillation (jacket temperature 65° C., 110 mbar pressure) untilapproximately 27 L of distillate had been removed. The remainingsolution in the vessel was cooled to afford(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine asa solution in toluene (33% w/w) (15.4 Kg, 97%). ¹H NMR (500 MHz, DMSO,27° C.) 0.98 (3H, d), 1.26 (3H, d), 1.30 (3H, d), 2.57-2.75 (3H, m),2.81 (1H, dd), 2.84-2.92 (1H, m), 6.97 (1H, t), 7.06 (1H, t), 7.11-7.22(1H, multiplet obscured by toluene signals), 7.34 (1H, d), 7.52 (1H, d),10.80 (1H, s).

Preparation of sodium{2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)methanesulfonate

2,6-Difluoro-4-bromobenzaldehyde (1000 g, 4.39 mol) and1,1-bis(di-tert-butylphosphino) ferrocene palladium dichloride (57.2 g;87.76 mmol) were charged to a 20 L vessel. Tetra-n-butylammoniumchloride (122 g, 438.97 mmol) was added followed by dimethylacetamide (5L). The vessel was purged with a stream of nitrogen gas.Diisopropylethylamine (1.5 L, 8.78 mol) was added to the vessel followedby methyl acrylate (0.435 L, 4.82 mol). The mixture was agitated andheated to 60° C. The mixture was held at this temperature for 20 hours.Ethyl acetate (10 L) was added to the mixture and the heating wasswitched off. Water (5 L) was added to the vessel. The stirred mixturewas cooled to 25° C. and stirring was continued for 10 minutes.Agitation was stopped and the layers were allowed to separate. Theaqueous layer was removed and discarded. The organic layer was washedsequentially with hydrochloric acid (2.2M, 6 L) and water (5 L).Phosphonics SPM32 Scavenger (1050 g, 1050 mol) was added to the vesseland the mixture was stirred for 3 days at 25° C. The solid material wasremoved by filtration. The cake was washed with ethanol (5 L) and thecombined filtrates were concentrated under reduced pressure to afford asolid. The solid was dissolved in ethanol (9 L) and the solution wasagitated in a 20 L vessel. The solution was heated to 50° C. A solutionof sodium bisulfite (460 g, 4.42 mol) in water (2.5 L) was added over 30minutes. A thick slurry resulted which was stirred for 4 hours at 50° C.The slurry was cooled to 20° C. over 2 hours. The solid was isolated byfiltration and the vessel and filter cake were washed with MTBE (2×3 L).The resulting solid was dried in vacuo to afford sodium{2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl)}(hydroxy)methanesulfonate(1035 g, 71%) as a light brown solid. ¹H NMR (500 MHz, DMSO, 27° C.) δ3.73 (3H, s), 5.32 (1H, d), 5.94 (1H, d), 6.76 (1H, d), 7.40 (2H, d),7.60 (1H, d).

Preparation of (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate

Under an atmosphere of nitrogen, sodium{2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)methanesulfonate(1.211 kg, 3.52 mol) was to a 100 L vessel followed by potassiumcarbonate (0.974 kg, 7.05 mol). Water (9.1 L) was added and the agitatorwas started. Ethyl acetate (9.1 L) was added. The mixture was agitatedat 25° C. for 5 hours. The agitator was stopped and the mixture wasallowed to stand for 14 hours at 25° C. The lower aqueous phase wasremoved and discarded. The upper organic phase was concentrated underreduced pressure to afford a pale brown solid. The solid was dried invacuo to afford (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate as abrown solid (608 g, 76%). ¹H NMR (500 MHz, DMSO, 27° C.) δ 3.77 (3H, s),6.94 (1H, d), 7.66 (1H, d), 7.71 (2H, d), 10.20 (1H, s).

Preparation of (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

Under an atmosphere of nitrogen, (E)-Methyl3-(3,5-difluoro-4-formylphenyl)acrylate (0.606 kg, 2.65 mol) and(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine(33% w/w solution in toluene, 2.0 kg, 2.65 mol) were charged to a 20 Lvessel followed by toluene (4.22 L). Acetic acid (304 ml, 5.31 mol) wasadded to the vessel. The mixture was agitated and heated to 80° C. Themixture was agitated at 80° C. overnight before being cooled to 20° C. Asolution of potassium carbonate (0.916 kg, 6.63 mol) in water (3.3 L)was added to the mixture. The mixture was stirred for 10 minutes beforethe agitator was switched off and the layers were allowed to separate.The aqueous layer was removed and discarded. Water (3.3 L) was chargedto the reactor. The mixture was agitated for 10 minutes then allowed tostand for 10 minutes. The lower aqueous phase was removed and theorganic layer was allowed to stand at room temperature overnight. Thebatch was heated to 80° C. Heptane (4.61 L) was added to the hotsolution over 35 minutes. The stirred mixture was held at approximately80° C. for 1 hour. The mixture was cooled to 30° C. over 2 hours duringwhich time the product crystallised. The slurry was stirred at 30° C.for 2.5 hours. The solid was isolated by filtration. The reactor vesselwalls were washed with heptane and the washings were used to wash thefilter cake. The solid was dried in vacuo to afford (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateas a pink solid (0.763 kg, 61%). ¹H NMR (400 MHz, DMSO, 27° C.) δ 1.06(3H, d), 1.13 (3H, d), 1.21 (3H, d), 2.35 (1H, dd), 2.58 (1H, dd),2.8-2.98 (2H, m), 3.44-3.61 (1H, m), 3.74 (3H, s), 5.24 (1H, s), 6.80(1H, d), 6.9-7.05 (2H, m), 7.19 (1H, d), 7.41 (1H, d), 7.50 (2H, d),7.63 (1H, d), 10.58 (1H, s). m/z: ES+ [M+H]+ 457.

Alternative Preparation of Example 1:(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

7.5M Sodium hydroxide (32.9 ml, 247.10 mmol) was added to a solution of(E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(11.28 g, 24.71 mmol) in THF (143 ml) and methanol (71.4 ml). Thereaction was stirred at room temperature for 4 h. The pH of the aqueouswas adjusted to ˜6.5 by addition of 2N HCl solution, then the solutionwas extracted with diethyl ether (3×150 ml). The combined organics weredried over Na₂SO₄ and concentrated. The crude product was purified byflash silica chromatography, elution gradient 0 to 20% methanol in DCMwhich afforded a yellow solid. Attempted trituration withacetone/heptane failed due to higher than expected solubility. Thesolvents were removed to give a yellow solid which was triturated inisohexane (50 ml) with a few drops of diethyl ether, the resulting solidwas filtered off and dried to give crude product (11.14 g) as a yellowpowder. The solid was dissolved in ethanol (100 ml), under nitrogen andin the dark. The solution was then evaporated to 5 mBar using a vacuumpump at 62° C. in the dark. This procedure was repeated twice and theresulting yellow glass scratched with a spatula into a fine powder andsubjected to 5 mBar using a vacuum pump at 62° C. for 60 min, to afforda yellow powder. The powder was then left in a vacuum oven over P205 at62° C. at 300 mBar overnight to afford the title product (9.77 g, 89%)as a pale yellow solid. ¹H NMR (400 MHz, DMSO, 27° C.) δ 1.07-1.16 (3H,m), 1.18-1.29 (6H, m), 2.39 (1H, dd), 2.62 (1H, dd), 2.92 (2H, dd), 3.56(1H, d), 5.26 (1H, s), 6.70 (1H, d), 7.02 (2H, dd), 7.22 (1H, d), 7.47(3H, dd), 7.58 (1H, d), 10.60 (1H, s), 12.60 (1H, s). m/z:ES+(ElectroSpray+) [M+H]+ 443.

The (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateused as starting material was prepared as follows:—

Preparation of 2-fluoro-2-methylpropan-1-ol

Lithium aluminium hydride (3.37 g, 88.56 mmol) was added portionwiseover 15 min to a cooled solution of ethyl 2-fluoro-2-methylpropanoate(9.9 g, 73.80 mmol) in diethyl ether (184 ml) at 0° C. The reaction wasstirred for 1 hr, then water (3.3 ml), followed by 15% NaOH solution(3.3 ml) and water (6.7 ml) were added sequentially. The suspension wasstirred for 15 min, then filtered and the solids washed with diethylether. The filtrate was evaporated to give 2-fluoro-2-methylpropan-1-ol(5.90 g, 87%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃, 27° C.) δ1.37 (6H, d), 3.56 (2H, d), OH not observed.

Alternative Preparation of 2-fluoro-2-methylpropyltrifluoromethanesulfonate

Trifluoromethanesulfonic anhydride (12.06 ml, 71.24 mmol), followed by2,6-lutidine (11.42 ml, 81.42 mmol) were added to a solution of2-fluoro-2-methylpropan-1-ol (6.25 g, 67.85 mmol) in DCM (146 ml) at−10° C. The reaction was stirred for 1 hr, then washed with 2N HCl(2×100 ml) and saturated NaHCO₃ solution (2×100 ml). The organic phasewas then dried over Na₂SO₄ and concentrated to give2-fluoro-2-methylpropyl trifluoromethanesulfonate (12.89 g, 85%) as ared oil.

¹H NMR (400 MHz, CDCl₃, 27° C.) δ 1.46 (6H, d), 4.41 (2H, d).

This intermediate may be purified by vacuum distillation. Analysis byDSC showed the material had the potential to self heat. For reasons ofprocess safety a wiped film evaporator or similar may be preferable to abatch distillation.

Alternative Preparation of(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine

2-Fluoro-2-methylpropyl trifluoromethanesulfonate (8.04 g, 35.87 mmol)was added to a solution of (R)-1-(1H-indol-3-yl)propan-2-amine (5.00 g,28.70 mmol) and N-ethyl-N-isopropylpropan-2-amine (7.44 ml, 43.04 mmol)in dioxane (50 ml). The reaction was heated to 90° C. for 3 h. Aftercooling to room temperature, the reaction was diluted with EtOAc (200ml) and washed with saturated. NaHCO₃ solution (2×100 ml). The aqueousphase was extracted with EtOAc (150 ml), then the combined organics weredried over MgSO₄ and concentrated. The crude product was purified byflash silica chromatography, elution gradient 100% EtOAc. Pure fractionswere evaporated to dryness to afford(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine(6.49 g, 91%) as a brown oil. ¹H NMR (400 MHz, CDCl₃, 27° C.) δ 1.14(3H, d), 1.31 (3H, d), 1.37 (3H, d), 1.94 (1H, s), 2.63-2.87 (3H, m),2.92 (1H, dd), 3.07 (1H, h), 7.07 (1H, d), 7.08-7.15 (1H, m), 7.16-7.24(1H, m), 7.37 (1H, d), 7.62 (1H, d), 8.04 (1H, s). m/z: ES+ [M+H]+ 249

Alternative Preparation of (E)-methyl3-(3,5-difluoro-4-formylphenyl)acrylate

4-Bromo-2,6-difluorobenzaldehyde (9.99 g, 45.20 mmol) and methylacrylate (6.14 ml, 67.81 mmol) were taken up in thoroughly degassed DMA(100 ml) and tri-o-tolylphosphine (1.376 g, 4.52 mmol), palladium(II)acetate (0.507 g, 2.26 mmol) and triethylamine (12.60 ml, 90.41 mmol)added. The reaction was stirred and heated to 80° C. for 6 hours. Thereaction mixture was cooled and filtered through a layer of celite, andwashed with methanol (50 ml). The crude product was pre-absorbed ontosilica and purified by suction chromatography eluting with 0-10% diethylether/dichloromethane. Fractions containing the desired product wereevaporated and triturated with diethyl ether (50 ml) to afford a yellowsolid which was triturated with water (50 ml) and dried under highvacuum at 50° C. to afford (E)-methyl3-(3,5-difluoro-4-formylphenyl)acrylate (8.85 g, 87%) as a yellow solid.¹H NMR (400 MHz, DMSO, 27° C.) δ 3.75 (3H, s), 6.93 (1H, d), 7.52-7.81(3H, m), 10.18 (1H, s). No mass ion observed in LCMS.

Alternative Preparation of (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

(E)-Methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (6.58 g, 29.09 mmol)was added to a suspension of(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine(6.02 g, 24.24 mmol) in toluene (51.1 ml) and acetic acid (2.78 ml,48.48 mmol). The reaction was heated to 80° C. for 5 hours. The reactionmixture was purified by ion exchange chromatography, using an SCX-2column. The desired product was eluted from the column using 7MNH₃/methanol and pure fractions were evaporated to dryness to afford abrown solid. The crude product was purified by flash silicachromatography, elution gradient 0 to 30% EtOAc in heptane. Purefractions were evaporated to dryness to afford the title product (7.52g, 68.0%) as a yellow solid. ¹H NMR (400 MHz, DMSO, 100° C.) δ 1.10 (3H,d), 1.12-1.31 (6H, m), 2.28-2.72 (2H, m), 2.84-3.09 (2H, m), 3.52-3.69(1H, m), 3.76 (3H, s), 5.30 (1H, s), 6.64 (1H, d), 6.9-7.11 (2H, m),7.21 (1H, d), 7.32 (2H, d), 7.42 (1H, d), 7.58 (1H, d), 10.14 (1H, s).m/z: ES+ [M+H]+ 457

Example 2(E)-3-(4-((1R,3R)-2-(2-Fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

7.5M Sodium hydroxide solution (0.983 ml, 7.37 mmol) was added to asolution of (E)-methyl3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(310 mg, 0.74 mmol) in methanol (5 ml). The mixture was stirred at 20°C. for 2 hours. The reaction mixture was purified by ion exchangechromatography, using an SCX-2 column. Fractions containing the desiredproduct were eluted from the column using 7M NH₃/methanol and purefractions were evaporated to dryness to afford a yellow solid. The crudeproduct was purified by preparative HPLC (Waters SunFire column, 5 μsilica, 50 mm diameter, 100 mm length), using decreasingly polarmixtures of water (containing 0.1% formic acid) and MeCN as eluents.Fractions containing the desired compound were evaporated to dryness andthen loaded onto SCX-2 column and eluted with 7N ammonia in methanol toafford the title product (63.0 mg, 21.02%) as a yellow solid. ¹H NMR(400 MHz, DMSO, 30° C.) δ 1.06 (3H, d), 1.30 (3H, d), 1.47 (3H, d),2.53-2.64 (2H, m), 2.79 (2H, s), 3.10 (1H, d), 5.08 (1H, s), 6.47 (1H,d), 6.98 (1H, t), 7.06 (1H, t), 7.19-7.37 (3H, m), 7.44 (1H, d), 7.56(1H, d), 7.63 (2H, d), 10.81 (1H, s), 12.30 (1H, s). m/z: ES+ [M+H]+407.

The (E)-methyl3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateused as starting material was prepared as follows:—

Preparation of (E)-methyl 3-(4-formylphenyl)acrylate

4-Bromobenzaldehyde (30 g, 162.15 mmol) and methyl acrylate (20.94 g,243.22 mmol) were taken up in thoroughly degassed DMA (300 ml) andtreated with tri-o-tolylphosphine (4.94 g, 16.21 mmol), palladium(II)acetate (1.820 g, 8.11 mmol) and triethylamine (45.2 ml, 324.29 mmol)and heated to 110° C. for 16 hours. The reaction appeared complete afterthis time. The reaction mixture was poured into water (4 L) and theresulting precipitate was filtered and dried. The solid waschromatographed on silica, eluting with 100% heptane to 30% EtOAc inheptane. Relevant fractions were combined and evaporated to dryness toafford a yellow solid product which was triturated with heptane,filtered and washed with cold heptane. The solid was dried to afford(E)-methyl 3-(4-formylphenyl)acrylate (25.6 g, 83%) as a yellowcrystalline product. ¹H NMR (400 MHz, DMSO, 30° C.) δ 3.75 (3H, s), 6.79(1H, d), 7.72 (1H, d), 7.93 (4H, s), 10.03 (1H, s). No mass ion observedin LCMS.

Preparation of (E)-methyl3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine(obtained as described in Example 1, preparation of starting materials)(450 mg, 1.81 mmol) and (E)-methyl 3-(4-formylphenyl)acrylate (345 mg,1.81 mmol) were dissolved in toluene (15 ml), acetic acid (5 ml) andmolecular sieves were added. The reaction was stirred at 110° C. for 16hours under nitrogen then cooled to room temperature. The crude productwas purified by ion exchange chromatography, using an SCX-2 column. Thedesired product was eluted from the column using 7M NH₃/methanol andpure fractions were evaporated to dryness to afford crude product. Thecrude product was purified by flash silica chromatography, elutiongradient 0 to 30% EtOAc in heptane. Pure fractions were evaporated todryness to afford (E)-methyl3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(317 mg, 41.6%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃, 30° C.) δ1.09 (3H, d), 1.30 (3H, d), 1.47 (3H, d), 2.48-2.78 (4H, m), 3.30 (1H,m), 3.79 (3H, s), 5.09 (1H, s), 6.40 (1H, d), 7.12 (1H, td), 7.17 (1H,td), 7.29 (1H, d), 7.34 (2H, d), 7.43 (2H, d), 7.54 (1H, d), 7.66 (2H,m). m/z: ES+ [M+H]+ 421.

Example 3(E)-3-(3,5-Difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

2M Sodium hydroxide (3.0 ml, 6.00 mmol) was added to a solution of(E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(275 mg, 0.60 mmol) in THF (1.5 ml)/methanol (1.5 ml). The reaction wasstirred at room temperature for 3 h. EtOAc (15 ml) and water (15 ml)were added, then the pH of the aqueous was adjusted to ˜7 by addition of2N HCl. The layers were separated and the aqueous was extracted withEtOAc (15 ml). The combined organics were dried over Na₂SO₄ andconcentrated. The crude product was purified by flash silicachromatography, elution gradient 0 to 10% methanol in DCM. Purefractions were evaporated to dryness to afford the title product (250mg, 94%) as a pale yellow solid. ¹H NMR (400 MHz, DMSO, 30° C.) δ 0.77(3H, d), 1.06 (3H, d), 1.93 (1H, m), 2.18 (1H, dd), 2.58 (1H, dd), 2.65(1H, dd), 2.84 (1H, dd), 3.35 (1H, dd), 4.32 (1H, d), 4.44 (1H, d), 5.16(1H, s), 6.67 (1H, d), 6.93-7.04 (2H, m), 7.21 (1H, d), 7.42 (1H, d),7.46 (2H, m), 7.54 (1H, d), 10.57 (1H, s), 12.51 (1H, s). m/z: ES+[M+H]+ 443.

The (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateused as starting material was prepared as follows:—

Preparation of (S)-3-fluoro-2-methylpropan-1-ol

N,N-Diethyl-1,1,2,3,3,3-hexafluoropropan-1-amine (18.25 ml, 100.57 mmol)was added dropwise to a solution of (R)-methyl3-hydroxy-2-methylpropanoate (9.25 ml, 83.81 mmol) in DCM (77 ml)(reaction warms to ˜40° C.). The reaction was stirred for 1 h at ambienttemperature, then warmed to reflux for 4 h, before cooling to roomtemperature overnight. The reaction mixture was poured onto ice, and thelayers separated. The aqueous was extracted with DCM (2×150 ml), thenthe combined organics were dried and carefully concentrated. The residuewas dissolved in THF (200 ml) and cooled in an ice-bath. Lithiumaluminium hydride (6.45 g, 167.61 mmol) was added in portions over 15min. The reaction was stirred at 0° C. for 1 h and warmed to roomtemperature for a further 1 h. After cooling in an ice-bath, thereaction was quenched by addition of water (7 ml), followed by 15% NaOH(7 ml), and finally water (21 ml). MgSO₄ was added until a granularsolid was formed. The solid was filtered through celite and the solidswashed with diethyl ether (50 ml). The filtrate was washed with 2N HCl(2×100 ml), then the organic phase was dried over Na₂SO₄ andconcentrated. The crude product was purified by flash silicachromatography, elution gradient 0 to 10% EtOAc in DCM. Pure fractionswere evaporated to dryness to afford (S)-3-fluoro-2-methylpropan-1-ol(6.42 g, 83%) as a straw coloured oil. ¹H NMR (400 MHz, CDCl₃, 30° C.) δ0.97 (3H, dd), 1.96-2.14 (1H, m), 3.64 (2H, d), 4.3-4.42 (1H, m),4.42-4.54 (1H, m), OH not observed.

Preparation of (S)-3-fluoro-2-methylpropyl trifluoromethanesulfonate

To a stirred solution of (S)-3-fluoro-2-methylpropan-1-ol (7.9 g, 85.77mmol) in DCM (140 ml) at 0° C. was added trifluoromethanesulfonicanhydride (17.31 ml, 102.92 mmol) dropwise followed by dropwise additionof 2,6-dimethylpyridine (11.95 ml, 102.92 mmol). The reaction mixturewas stirred at 0° C. for 45 min and room temperature for 30 min. Thereaction mixture was diluted with DCM (60 ml), and washed sequentiallywith 1M HCl (3×100 ml), saturated sodium bicarbonate solution (100 ml)and saturated brine (50 ml). The organic layer was dried over Na₂SO₄,filtered and evaporated to near dryness. The solution was filteredthrough a plug of silica and washed through with DCM (50 ml) andevaporated to give (S)-3-fluoro-2-methylpropyl trifluoromethanesulfonate(14.38 g, 74.8%) as a brown oil. ¹H NMR (400 MHz, CDCl₃, 27° C.) δ 1.09(3H, dd), 2.24-2.44 (1H, m), 4.30 (0.5H, dd), 4.37-4.46 (1H, m), 4.52(2.5H, tt).

Preparation of(S)—N—((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amine

(S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (666 mg, 2.97mmol) was added to a solution of (R)-1-(1H-indol-3-yl)propan-2-amine(470 mg, 2.7 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.700 ml, 4.05mmol) in 1,4-dioxane (6.05 ml). The reaction was stirred at roomtemperature for 1 h. The reaction mixture was diluted with EtOAc (20 ml)and washed with water (20 ml). The aqueous was extracted with EtOAc(2×20 ml), then the combined organics were dried (MgSO₄) andconcentrated. The crude product was purified by flash silicachromatography, elution gradient 0 to 10% methanol in DCM. Purefractions were evaporated to dryness to afford(S)—N—((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amine(590 mg, 88%) as a brown oil. ¹H NMR (400 MHz, CDCl₃, 30° C.) δ 0.86(3H, dd), 1.20 (3H, d), 1.94-2.11 (1H, m), 2.64-2.74 (2H, m), 2.85-2.98(2H, m), 3.05-3.15 (1H, m), 4.13-4.39 (2H, m), 7.09 (1H, d), 7.12 (2H,ddd), 7.20 (1H, ddd), 7.33-7.41 (1H, m), 7.56-7.65 (1H, m), 8.10 (1H,s). m/z: ES+ [M+H]+ 249

Preparation of (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

Acetic acid (2.0 ml) was added to a solution of(S)—N—((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amine(273 mg, 1.10 mmol) and (E)-methyl3-(3,5-difluoro-4-formylphenyl)acrylate (obtained as described inExample 1, preparation of starting materials) (226 mg, 1 mmol) intoluene (8.0 ml). The reaction was warmed to 95° C. for 2.5 h. Thevolatiles were removed under vacuum, then the residue was passed throughan SCX-2 column. The column was then eluted with 7M NH₃/methanol toliberate the product. The filtrate was concentrated and the crudeproduct was purified by flash silica chromatography, elution gradient 0to 10% methanol in DCM. Pure fractions were evaporated to dryness toafford (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(282 mg, 61.8%) as a pale yellow solid. ¹H NMR (400 MHz, CDCl₃, 30° C.)δ 0.82 (3H, d), 1.12 (3H, d), 1.81-1.99 (1H, m), 2.24 (1H, ddd), 2.64(1H, ddd), 2.71 (1H, dd), 2.93-3.01 (1H, ddd), 3.42 (1H, dq), 3.81 (3H,s), 4.25-4.39 (1H, m), 4.38-4.53 (1H, m), 5.20 (1H, s), 6.39 (1H, d),6.99 (2H, m), 7.06-7.16 (2H, m), 7.21-7.25 (1H, m), 7.52 (3H, m). m/z:ES+ [M+H]+ 457.

Example 4(E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

7.5M Sodium hydroxide solution (0.904 ml, 6.78 mmol) was added to asolution of (E)-methyl3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(285 mg, 0.68 mmol) in methanol (3 ml). The mixture was stirred at 20°C. for 2 hours. The reaction mixture was purified by ion exchangechromatography, using an SCX-2 column. Fractions containing the desiredproduct were eluted from the column using 7M NH₃/methanol and purefractions were evaporated to dryness to afford a yellow solid. The crudeproduct was purified by preparative LCMS (Phenomenex Gemini-NX axia PrepC18 OBD column, 5 μ silica, 50 mm diameter, 100 mm length), usingdecreasingly polar mixtures of water (containing 1% NH₃) and MeCN aseluents. Fractions containing the desired compound were evaporated todryness to afford the title product (80 mg, 29.0%) as a yellow solid. ¹HNMR (500 MHz, DMSO, 33° C.) δ 0.87 (3H, d), 1.04 (3H, d), 1.91-2.27 (2H,m), 2.50 (1H, p), 2.57-2.75 (2H, m), 3.13 (1H, s), 4.51 (2H, dd), 4.86(1H, s), 6.47 (1H, d), 6.92-6.99 (1H, m), 7-7.09 (1H, m), 7.15-7.35 (3H,m), 7.35-7.5 (2H, m), 7.57 (2H, d), 10.64 (1H, d), CO₂H not observed.m/z: ES+ [M+H]+ 407.

The (E)-methyl3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateused as starting material was prepared as follows:—

Preparation of (E)-methyl3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

(E)-Methyl 3-(4-formylphenyl)acrylate (obtained as described in Example2, preparation of starting materials) (18.56 g, 97.57 mmol) was added toa stirred solution of (R)-1-(1H-indol-3-yl)propan-2-amine (17 g, 97.57mmol) in acetic acid (250 ml) at 23° C. under nitrogen. The resultingsolution was stirred at 80° C. for 2 hours. The reaction mixture wasevaporated to dryness and redissolved in DCM (500 ml), and washedsequentially with saturated NaHCO₃ (300 ml×2), 2M NaOH (aq) (300 ml),water (300 ml), and saturated brine (300 ml). The organic layer wasdried over MgSO₄, filtered and evaporated to afford crude product. Thecrude product was purified by flash silica chromatography, elutiongradient 1 to 7% methanol in DCM. Pure fractions were evaporated todryness to afford (E)-methyl 3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(25.1 g, 74.3%) as a beige foam. The product was mostly cis isomer,containing about 12% trans isomer which was inseparable. ¹H NMR (400MHz, DMSO, 30° C.) δ 1.25 (3H, d), 2.37-2.48 (1H, m), 2.74 (1H, d), 3.12(1H, s), 3.73 (3H, s), 5.18 (1H, s), 6.64 (1H, d), 6.97 (2H, dd), 7.19(1H, d), 7.36-7.46 (3H, m), 7.64-7.75 (3H, m), 10.19 (1H, s), no NHobserved. m/z: ES+ [M+H]+ 347.

Preparation of (E)-methyl3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

(E)-Methyl3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(35 g, 101.03 mmol), 3-bromoprop-1-ene (9.62 ml, 111.14 mmol) andN-ethyl-N-isopropylpropan-2-amine (19.36 ml, 111.14 mmol) were suspendedin acetonitrile (160 ml), nitrogen was bubbled through for 5 min andthen the mixture was sealed into a microwave tube. The reaction washeated to 140° C. for 3.5 hours in the microwave reactor and cooled toroom temperature.

The reaction mixture was evaporated to dryness and redissolved in DCM(100 ml), and washed sequentially with 1M citric acid (100 ml), water(100 ml), and saturated brine (100 ml). The organic layer was dried overMgSO₄, filtered and evaporated to afford crude product. The crudeproduct was purified by flash silica chromatography, elution gradient 0to 20% EtOAc in heptane. Pure fractions were evaporated to dryness toafford a 50:50 mixture of (E)-methyl3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate:(E)-methyl3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(10.00 g, 25.6%) as a pale yellow solid. m/z: ES+ [M+H]+ 387.

Preparation of (E)-methyl3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

Trifluoroacetic acid (5.59 ml, 75.29 mmol) was added to (E)-methyl3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(9.7 g, 25.10 mmol) in DCM (100 ml) at 20° C. under nitrogen. Theresulting solution was stirred at 20° C. for 3 days. The reactionmixture was diluted cautiously with saturated NaHCO₃ solution (250 ml),and the DCM layer washed sequentially with water (250 ml) and saturatedbrine (250 ml). The organic layer was dried over MgSO₄, filtered andevaporated to afford crude product. The crude product was purified byflash silica chromatography, elution gradient 10 to 20% EtOAc inheptane. Fractions were evaporated to dryness to afford a 65:35 mixtureof (E)-methyl3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate:(E)-methyl3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(7.99 g, 82%) as a pale yellow solid. m/z: ES+ [M+H]+ 387

Preparation of (E)-methyl3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

7 Separate batches of 65:35 (E)-methyl3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate:(E)-methyl3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(2.00 g, 5.17 mmol) were reacted as follows.

(E)-methyl3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(2.00 g, 5.17 mmol) and chlorotris(triphenylphosphine)rhodium(I)(Wilkinson's catalyst) (2.346 g, 2.54 mmol) were suspended inacetonitrile (12 ml) and water (2.4 ml) and nitrogen was bubbled throughfor 5 min before being sealed into a microwave tube. The reaction washeated to 100° C. for 60 min in the microwave reactor and cooled to roomtemperature. The reaction mixtures were combined and evaporated todryness and redissolved in DCM (200 ml) and saturated NaHCO₃ solution(200 ml) added. The organic layer was washed sequentially with water(200 ml) and saturated brine (200 ml) before being dried over MgSO₄,filtered and evaporated to afford crude product. The crude product waspurified by flash silica chromatography, elution gradient 0 to 5%methanol in DCM. Pure fractions were evaporated to dryness to afford(E)-methyl3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(7.02 g, 55.9%) as a pale yellow solid. ¹H NMR (400 MHz, DMSO, 30° C.) δ1.14 (3H, d), 2.27-2.4 (1H, m), 2.81 (1H, dd), 2.94-3.05 (1H, m), 3.72(3H, s), 5.19 (1H, s), 6.60 (1H, d), 6.94-7 (1H, m), 7.01-7.09 (1H, m),7.26 (3H, d), 7.43 (1H, d), 7.59-7.68 (3H, m), 10.70 (1H, s), NH notobserved. m/z: ES+ [M+H]+ 347

Preparation of (E)-methyl3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

(S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained asdescribed in Example 3, preparation of starting materials) (291 mg, 1.30mmol) was added to a solution of (E)-methyl3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(300 mg, 0.87 mmol) and N,N-diisopropylethylamine (0.226 ml, 1.30 mmol)in 1,4-dioxane (5 ml). The mixture was stirred at 90° C. for 1 hour thenthe mixture was evaporated to dryness and the residue was partitionedbetween DCM (30 ml) and water (30 ml). The aqueous layer was extractedwith DCM (30 ml) and the extracts combined with the organic layer. Thecombined extracts were filtered through a phase-separating paper andevaporated. The residue was purified by flash silica chromatography,elution solvent 15% EtOAc in heptane. Pure fractions were evaporated todryness to afford (E)-methyl3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(314 mg, 86%) as an off-white solid. ¹H NMR (400 MHz, DMSO, 30° C.) δ0.87 (3H, d), 1.06 (3H, d), 1.9-2.28 (2H, m), 2.55-2.8 (3H, m),2.97-3.21 (1H, m), 3.72 (3H, s), 4.31-4.69 (2H, m), 4.88 (1H, s), 6.60(1H, dd), 6.98 (1H, t), 7.04 (1H, t), 7.17-7.35 (3H, m), 7.44 (1H, d),7.55-7.76 (3H, m), 10.65 (1H, s). m/z: ES+ [M+H]+ 421.

Example 5(E)-3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 1)*

Sodium hydroxide (184 mg, 4.61 mmol) was added to (E)-methyl3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) (217 mg, 0.46 mmol) in THF (1 ml)/methanol (1 ml). Theresulting solution was stirred at 20° C. for 16 hours. The reaction wasdiluted with water (10 ml) and the pH was adjusted to 7 by the additionof 2N HCl. The solution was extracted with EtOAc (2×20 ml). The combinedorganics were dried over Na₂SO₄ and concentrated. The crude product waspurified by flash silica chromatography, elution gradient 0 to 50% EtOAcin heptane. The pure fractions were evaporated to give a crude material.The crude product was triturated using a diethyl ether/isohexane mixtureto give the title product (53.0 mg, 25.2%) as a yellow solid. ¹H NMR(400 MHz, DMSO, 27° C.) δ 0.54 (3H, d), 1.06 (3H, s), 1.34 (3H, s), 2.14(1H, dd), 2.66 (1H, d), 2.83 (1H, d), 3.03 (1H, dd), 4.21 (1H, t), 4.33(1H, t), 5.12 (1H, s), 6.73 (1H, d), 7.01 (2H, dtd), 7.14-7.28 (1H, m),7.43 (1H, d), 7.54 (2H, s), 7.59 (1H, d), 10.50 (1H, s), 12.61 (1H, s),CO₂H not observed. m/z: ES+ [M+H]+ 457 * Stereochemistry inferred to be(R) at the undefined centre by analogy with other examples, ie compoundinferred to be:(E)-3-(3,5-difluoro-4-(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

The3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) used as starting material was prepared as follows:—

Preparation of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate

1-(1H-Indol-3-yl)-2-methylpropan-2-amine (807 mg, 4.29 mmol) and(E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (obtained asdescribed in Example 1, preparation of starting materials) (970 mg, 4.29mmol) were combined in acetic acid (15 ml) and the mixture heated to 80°C. for 2 hours. The reaction mixture was purified by ion exchangechromatography, using an SCX-2 column. The desired product was elutedfrom the column using 2M NH₃ in methanol and product-containingfractions were evaporated to dryness to afford (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate(1610 mg, 95%) as a yellow foam. ¹H NMR (500 MHz, DMSO, 20° C.) δ 1.15(3H, s), 1.27 (3H, s), 2.23 (1H, s), 2.61 (2H, s), 3.75 (3H, s), 5.46(1H, s), 6.84 (1H, d), 6.97 (2H, dtd), 7.18 (1H, d), 7.39 (1H, d), 7.58(2H, s), 7.67 (1H, d), 10.60 (1H, s). m/z: ES− [M−H]− 395.

Preparation of (E)-methyl3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1)

(S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained asdescribed in Example 3, preparation of starting materials) (0.339 g,1.51 mmol) was added to a solution of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate(0.3 g, 0.76 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.458 ml, 2.65mmol) in 1,4-dioxane (2 ml). The stirring was continued for 24 hoursthen the volatiles were removed under vacuum and the crude product waspurified by flash silica chromatography, elution gradient 0 to 25% EtOAcin heptane. Pure fractions were evaporated to dryness to afford(E)-methyl3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(0.202 g, 36%) as a white solid. The material was combined with anotherbatch (0.36 g) and purified by preparative HPLC (Chiralpak IA column, 20μm silica, 20 mm diameter, 250 mm length), Heptane:IPA 70:30 at 80ml/min (4 injections). Fractions containing the desired compounds wereevaporated to yield (E)-methyl3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1, first eluted, 217 mg) and (E)-methyl3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 2, second eluted, 165 mg). Analysis was done on Chiralpak IAcolumn, 5 μm silica, 4.6 mm diameter, 50 mm length, Heptane:IPA 70:30 at2 ml/min.

¹H NMR (400 MHz, DMSO, 30° C.) δ 0.50 (3H, d), 1.02 (3H, s), 1.30 (3H,s), 2.11 (1H, dd), 2.62 (2H, d), 2.80 (1H, d), 2.99 (1H, dd), 3.74 (3H,s), 4.09-4.23 (1H, m), 4.29 (1H, d), 5.09 (1H, s), 6.81 (1H, d), 6.97(2H, dt), 7.16 (1H, d), 7.39 (1H, d), 7.53 (2H, d), 7.64 (1H, d), 10.44(1H, s). m/z: ES+ [M+H]+ 471.

Example 6(E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 1)*

2M Sodium hydroxide (1.6 ml, 3.20 mmol) was added to a solution of(E)-methyl3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) (148 mg, 0.31 mmol) in THF (0.8 ml)/methanol (0.8 ml). Thereaction was stirred at room temperature for 3 h. EtOAc (15 ml) andwater (15 ml) were added, and the pH of the aqueous was adjusted to ˜7by addition of 2N HCl. The layers were separated, and the aqueous wasextracted with EtOAc (15 ml). The combined organics were dried overNa₂SO₄ and concentrated. The crude product was purified by flash silicachromatography, elution gradient 25 to 100% EtOAc in heptane. Purefractions were evaporated to dryness to afford the title product(isomer 1) (124 mg, 86%) as a pale yellow solid. ¹H NMR (400 MHz, CDCl₃,30° C.) δ 1.03 (3H, d), 1.08 (3H, s), 1.16 (3H, d), 1.35 (3H, s), 2.56(1H, dd), 2.66 (1H, d), 3.02 (1H, d), 3.17 (1H, dd), 5.24 (1H, s), 6.39(1H, d), 7.00 (2H, d), 7.05-7.16 (2H, m), 7.20 (1H, dd), 7.28 (1H, s),7.49 (1H, dd), 7.61 (1H, d), CO₂H not observed. m/z: ES+ [M+H]+ 457. *Stereochemistry inferred to be (R) at the undefined centre by analogywith other examples.

The (E)-methyl3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) used as starting material was prepared as follows:—

Preparation of (E)-methyl3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1)

2-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as describedin Example 1, preparation of starting materials) (679 mg, 3.03 mmol) wasadded to a solution of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate(obtained as described in Example 5, preparation of starting materials)(600 mg, 1.51 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.915 ml,5.30 mmol) in 1,4-dioxane (2.5 ml). The reaction was stirred at roomtemperature for 1 h. The reaction was then heated to 105° C. for 88hours. The volatiles were removed under vacuum and the crude product waspurified by flash silica chromatography, elution gradient 0 to 50% EtOAcin heptane. Pure fractions were evaporated to dryness to afford(E)-methyl3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(430 mg, 60.4%) as a white solid. The racemic product was purified bypreparative HPLC (Chiralpak AD column, 20 μm silica, 50 mm diameter, 250mm length), Heptane:Ethanol 90:10 90 ml/min. Fractions containing thedesired compounds were evaporated to dryness to afford (E)-methyl3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1, first eluted, 149 mg, 34.6%) and (E)-methyl3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 2, second eluted, 143 mg, 33.3%) as cream coloured solids. ¹HNMR (400 MHz, CDCl₃, 30° C.) δ 1.01 (3H, d), 1.08 (3H, s), 1.15 (3H, d),1.34 (3H, s), 2.55 (1H, dd), 2.66 (1H, d), 2.98-3.06 (1H, m), 3.17 (1H,dd), 3.80 (3H, s), 5.23 (1H, s), 6.38 (1H, d), 6.97 (2H, d), 7.07-7.12(2H, m), 7.17-7.22 (1H, m), 7.32 (1H, s), 7.46-7.5 (1H, m), 7.52 (1H,d). m/z ES− [M−H]− 469.

Example 7(E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 1)*

2M Sodium hydroxide solution (20.42 ml, 40.85 mmol) was added to asolution of (E)-methyl3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) (3.55 g, 8.17 mmol) in methanol (100 ml), THF (100 ml) andwater (75 ml). The mixture was heated at 40° C. for 16 hours. Themixture was diluted with water (100 ml) and concentrated to a volumesuch that the organic solvents had been removed. The resulting aqueoussolution was acidified to pH 6 with 2M HCl. The resulting aqueoussuspension was extracted with DCM (500 ml) (adding brine to helpseparate the emulsion which formed), filtered through a phase-separatingpaper, dried over MgSO₄ then filtered through celite and evaporated toafford ˜3.5 g of a pale yellow solid. The crude product was treated withdiethyl ether/DCM (1:1, 150 ml) and sonicated. The fine suspension whichformed was passed through a pad of silica (˜100 g) and the silica waseluted with diethyl ether (˜2 L). Product-containing fractions werecombined, evaporated and dried under vacuum at 50° C. to afford thetitle product (2.305 g, 64.5%) as a beige solid. ¹H NMR (400 MHz, DMSO,30° C.) δ 0.52 (3H, d), 1.03 (3H, s), 1.05-1.16 (1H, m), 1.23 (3H, s),2.21 (1H, dd), 2.65 (1H, d), 2.84 (1H, d), 2.89 (1H, dd), 4.19 (1H,ddd), 4.31 (1H, ddd), 4.66 (1H, s), 6.50 (1H, d), 6.93 (1H, ddd), 6.98(1H, ddd), 7.18 (1H, d), 7.38 (2H, d), 7.40 (1H, d), 7.58 (1H, d), 7.63(2H, d), 10.18 (1H, s), 12.30 (1H, s). m/z: ES+ [M+H]+ 421. *Stereochemistry inferred to be (R) at the undefined centre by analogywith other examples.

The product (9.0 g, 21.40 mmol) was slurried in acetonitrile (150 ml)under nitrogen in the dark for 1 hour in a stoppered 250 ml roundbottomed flask. The mixture was stirred over the weekend at roomtemperature then filtered and washed with cold acetonitrile (60 ml) toafford a white solid which was dried under high vacuum at 40° C. for 5hours to yield crystalline form A of the title product (7.81 g, 87%).

An XRPD trace of Crystalline form A includes the following peaks and isshown in FIG. 1.

2-Theta ° % 4.48 100 10.76 42.2 9.88 21.4 6.13 20.8 13.41 18.9 14.0118.2 14.31 14.7 18.46 13.2 7.92 12.2 4.76 9.3

The (E)-methyl3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) used as starting material was prepared as follows:—

Preparation of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(racemate)

(E)-Methyl 3-(4-formylphenyl)acrylate (41.8 g, 219.62 mmol) (obtained asdescribed in Example 2, preparation of starting materials) was added inone portion to 1-(1H-indol-3-yl)-2-methylpropan-2-amine (43.8 g, 219.62mmol) in acetic acid (314 ml) under nitrogen. The resulting solution wasstirred at 80° C. for 5 hours. The reaction mixture was concentrated invacuo. Toluene (200 ml) was added and the residue evaporated to dryness.The azeotrope treatment was repeated twice more to give a brown solid.This was stirred in 1:1 EtOAc/heptane (500 ml) for 30 min beforefiltering and washing with 1:1 EtOAc/heptane. The compound was air driedto give a white solid. The crude material was suspended in 2-methyltetrahydrofuran (750 ml), and saturated sodium bicarbonate solution wasadded over 10 min to the stirred mixture (effervescence), the mixturewas stirred until the material dissolved and the aqueous phase remainedbasic. The phases were separated and the organic phase washed withbrine, dried over MgSO₄, filtered and concentrated in vacuo to give apale yellow foam (˜78 g). The material was dissolved in diethyl ether(200 ml) and concentrated to dryness (repeated twice). On the secondaddition a proper solid was obtained. This was stirred in diethyl etherand evaporated to dryness to give (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(racemate) (73.6 g, 89%). ¹H NMR (400 MHz, CDCl₃, 30° C.) δ 1.26 (3H,s), 1.35 (3H, s), 1.42 (1H, br s), 2.69-2.82 (2H, m), 3.80 (3H, s), 5.12(1H, s), 6.41 (1H, d), 7.06-7.16 (2H, m), 7.21 (1H, dd), 7.37 (2H, d),7.46-7.54 (4H, m), 7.67 (1H, d). m/z: ES+ [M+H]+ 361.

Preparation of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1)

(E)-Methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(racemate) (65 g) was purified in seven injections as follows.

The racemic material was purified by preparative HPLC (Chiralpak ODcolumn, 20 μm silica, 100 mm diameter, 250 mm length), Heptane:IPA50:50. Fractions containing the desired compounds were evaporated todryness to afford (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1, first eluted, 30.3 g, 93%) and (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 2, second eluted, 28.2 g, 86%). ¹H NMR (400 MHz, CDCl₃, 30° C.)δ 1.27 (3H, s), 1.36 (3H, s), 2.69-2.82 (2H, m), 3.80 (3H, s), 5.14 (1H,s), 6.43 (1H, d), 7.12 (2H, pd), 7.2-7.24 (1H, m), 7.39 (3H, d), 7.51(3H, d), 7.68 (1H, d), NH not observed. m/z: ES+ [M+H]+ 361.

Preparation of (E)-methyl3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1)

(S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained asdescribed in Example 3, preparation of starting materials) (5.32 g,21.36 mmol) was added to a solution of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) (3.5 g, 9.71 mmol) and N-ethyl-N-isopropylpropan-2-amine(6.34 ml, 36.41 mmol) in 1,4-dioxane (17.5 ml). The mixture was stirredat 22° C. for 3 days. The mixture was evaporated and the residue waspartitioned between DCM (150 ml) and water (150 ml). The aqueous layerwas extracted with DCM (50 ml) and the extracts combined with theorganic layer. The combined extracts were filtered through aphase-separating paper and evaporated. The residue was purified by flashsilica chromatography, elution solvent 15% EtOAc in heptane. Fractionscontaining significant amounts of product began to form crystals; thetubes were agitated to encourage further crystallisation. The crystalswere collected by filtration and washed with a small amount of 15% EtOAcin heptane to afford (E)-methyl3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) (2.91 g, 69.0%) as a white crystalline solid. Liquors fromthe crystallisation and other product-containing fractions were combinedand evaporated. The residue was recrystallised from EtOAc/heptane toafford more3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) as a white crystalline solid (635 mg, 15.1%). ¹H NMR (400MHz, CDCl₃, 30° C.) δ 0.53 (3H, d), 0.95-1.07 (1H, m), 1.09 (3H, s),1.32 (3H, s), 2.16 (1H, dd), 2.66 (1H, d), 2.94 (1H, d), 2.97 (1H, d),3.80 (3H, s), 4.14 (1H, ddd), 4.31 (1H, ddd), 4.59 (1H, s), 6.42 (1H,d), 7.05-7.11 (2H, m), 7.13 (1H, s), 7.17 (1H, dd), 7.35 (2H, d), 7.45(2H, d), 7.50 (1H, dd), 7.67 (1H, d). m/z: ES+ [M+H]+ 435.

Example 8(E)-3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 1)*

2M Sodium hydroxide solution (0.782 ml, 1.56 mmol) was added to asolution of (E)-methyl3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) (68 mg, 0.16 mmol) in methanol (5 ml), THF (5.00 ml) andwater (5.00 ml) and the mixture stirred for 40 hours at 22° C. Themixture was concentrated to a volume such that all of the organicsolvent had been removed and was acidified to pH6 with 2M HCl.Concentrated aqueous ammonia (2 drops) was added, followed by methanol(˜2 ml), giving a pale yellow solution. The crude product was purifiedby preparative HPLC (Waters XBridge Prep C18 OBD column, 5 silica, 50 mmdiameter, 100 mm length), using decreasingly polar mixtures of water(containing 1% NH₃) and MeCN as eluents. Fractions containing thedesired compound were evaporated to dryness to afford the title product(isomer 1) (50.0 mg, 76%) as a yellow solid. ¹H NMR (500 MHz, DMSO, 30°C.) δ 0.93 (3H, s), 1.19 (3H, d), 1.23 (3H, s), 1.51 (3H, d), 2.56-2.64(2H, m), 2.75 (1H, d), 3.04 (1H, dd), 5.06 (1H, s), 6.40 (1H, d), 7.03(1H, ddd), 7.09-7.15 (2H, m), 7.35 (1H, dd), 7.44-7.51 (5H, m), 10.87(1H, s), CO₂H not observed. m/z: ES+ [M+H]+ 421. * Stereochemistryinferred to be (R) at the undefined centre by analogy with otherexamples.

The (E)-methyl3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1) used as starting material was prepared as follows:—

Preparation of (E)-methyl3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1)

2-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as describedin Example 1, preparation of starting materials) (389 mg, 1.73 mmol) wasadded to a solution of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(racemate) (obtained as described in Example 7, preparation of startingmaterials) (250 mg, 0.69 mmol) and N,N-diisopropylethylamine (0.453 ml,2.60 mmol) in 1,4-dioxane (1.25 ml). The mixture was stirred at 95° C.for 64 hours and then partitioned between DCM (30 ml) and water (30 ml).The aqueous layer was extracted with DCM (20 ml) and the extractscombined with the organic layer. The combined extracts were filteredthrough a phase-separating paper and evaporated. The residue waspurified by flash silica chromatography, elution solvent 15% EtOAc inheptane. Pure fractions were evaporated to dryness to afford (E)-methyl3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(racemate) (178 mg, 59.1%) as a beige solid. The racemic product waspurified by preparative HPLC (Chiralpak AD column, 20 μm silica, 50 mmdiameter, 250 mm length), Heptane:Ethanol: 80:20. Fractions containingthe desired compounds were evaporated to dryness to afford (E)-methyl3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 1, first eluted, 70 mg) and (E)-methyl3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(isomer 2, second eluted, 66 mg). ¹H NMR (400 MHz, CDCl₃, 30° C.) δ 1.00(3H, s), 1.13 (3H, d), 1.20 (3H, s), 1.44 (3H, d), 2.57-2.78 (3H, m),2.93 (1H, dd), 3.80 (3H, s), 5.05 (1H, s), 6.41 (1H, d), 7.13 (1H, ddd),7.18 (1H, ddd), 7.32 (1H, d), 7.43 (2H, d), 7.51 (2H, d), 7.54 (1H, d),7.64 (1H, s), 7.67 (1H, d). m/z: ES+ [M+H]+ 435.

Example 9(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 1)*

2M Sodium hydroxide (3.31 ml, 6.63 mmol) was added to a solution of(E)-methyl3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(600 mg, 1.33 mmol) (mixture of diastereoisomers) in methanol (5 ml),and THF (20 ml) and the mixture stirred at ambient temperature for 4hours. The mixture was concentrated to a volume such that all of theorganic solvent had been removed, diluted with water (50 ml), acidifiedwith dilute HCl to pH 6 and extracted with ethyl acetate (2×50 ml). Theextracts were combined and evaporated under reduced pressure. Theresidue was purified by preparative HPLC (Chiralpak IA column, 20 μmsilica, 20 mm diameter, 250 mm length), Heptane:IPA 90:10, 0.2% aceticacid at 80 ml/min. Fractions containing the desired compounds werecombined and analysed to yield(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 1, first eluted, 130 mg, 22.36%) and(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (isomer 2, second eluted, 30.0 mg, 5.16%).

¹H NMR (500 MHz, DMSO, 30° C.) δ 0.49 (3H, d), 1.03 (3H, s), 1.14-1.21(1H, m), 1.28 (3H, s), 2.17 (1H, dd), 2.59-2.71 (1H, m), 2.8-2.99 (2H,m), 4.09-4.34 (2H, m), 4.99 (1H, s), 6.59 (1H, d), 6.86-7.07 (2H, m),7.1-7.19 (1H, m), 7.18-7.29 (1H, m), 7.36-7.49 (2H, m), 7.49-7.66 (2H,m), 10.31 (1H, s), CO₂H not observed. m/z: ES+ [M+H]+ 439. *Stereochemistry inferred to be (R) at the undefined centre by analogywith other examples.

The (E)-methyl3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(mixture of diastereomers) used as starting material was prepared asfollows:—

Preparation of (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate

4-Bromo-2-fluorobenzaldehyde (20.88 g, 102.87 mmol) and methyl acrylate(13.98 ml, 154.30 mmol) were taken up in thoroughly degassed DMA (150ml) and tri-o-tolylphosphine (3.13 g, 10.29 mmol), palladium(II) acetate(1.155 g, 5.14 mmol) and triethylamine (28.7 ml, 205.74 mmol) added. Thereaction was stirred and heated to 100° C. for 16 hours. Moretri-o-tolylphosphine (3.13 g, 10.29 mmol) and palladium(II) acetate(1.155 g, 5.14 mmol) were added and the reaction mixture was heated to110° C. for a further 2 hours. Water (1 L) was added and the reactionmixture extracted with DCM (2×500 ml). Combined organics were dried(MgSO₄), filtered and evaporated to give a brown solid. The crudeproduct was purified by flash silica chromatography, elution gradient 0to 25% EtOAc in heptane. Pure fractions were evaporated to dryness toafford (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate (15.20 g, 71.0%)as a yellow solid.

¹H NMR (400 MHz, CDCl₃, 30° C.) δ 3.83 (3H, s), 6.53 (1H, d), 7.31 (1H,dd), 7.41 (1H, d), 7.65 (1H, d), 7.79-8 (1H, m), 10.25-10.41 (1H, m). Nomass ion observed in LCMS.

Preparation of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluorophenyl)acrylate(racemate)

1-(1H-Indol-3-yl)-2-methylpropan-2-amine (1 g, 5.31 mmol) and (E)-methyl3-(3-fluoro-4-formylphenyl)acrylate (1.106 g, 5.31 mmol) in acetic acid(15 ml) were stirred at 80° C. for 2 hours under nitrogen. The crudeproduct was purified by ion exchange chromatography, using an SCX-2column. The desired product was eluted from the column using 7MNH₃/methanol and pure fractions were evaporated to dryness to afford(E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluorophenyl)acrylate(racemate) (2.000 g, 99%). ¹H NMR (400 MHz, DMSO, 30° C.) δ 1.14 (3H,s), 1.27 (3H, s), 2.52-2.74 (2H, m), 3.74 (3H, s), 5.12 (1H, s), 6.69(1H, d), 6.86-7.05 (2H, m), 7.20 (1H, d), 7.25-7.33 (2H, m), 7.39 (1H,d), 7.73 (1H, d), 7.85 (1H, t), 10.29 (1H, s), NH not observed. m/z: ES+[M+H]+ 379.

Preparation of (E)-methyl3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(mixture of diastereomers)

(S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained asdescribed in Example 3, preparation of starting materials) (1.259 g,5.62 mmol) was added to a solution of (E)-methyl3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluorophenyl)acrylate(racemate) (850 mg, 2.25 mmol) and N-ethyl-N-isopropylpropan-2-amine(1.467 ml, 8.42 mmol) in 1,4-dioxane (5 ml). The mixture was heated at60° C. for 4 hours, then stirred at ambient temperature for 12 hours.The mixture was partitioned between ethyl acetate (25 ml) and water (25ml). The aqueous layer was extracted with ethyl acetate (2×25 ml) andthe extracts combined with the organic layer. The combined extracts wereevaporated under vacuum. The residue was purified by flash silicachromatography, elution solvent 10% EtOAc in heptane. Pure fractionswere evaporated to dryness to afford (E)-methyl3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(mixture of diastereoisomers) (600 mg, 59.0%). m/z: ES+ [M+H]+ 453.

Example 10(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoicacid

Methyl(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate(4.70 g, 10.27 mmol) was dissolved in iPrOH (42.8 ml) and 5M sodiumhydroxide solution (6.16 ml, 30.82 mmol) was added in one portion, thereaction was then stirred at room temperature for 4 hours. Water wasadded (100 ml) and the pH was brought to ˜5 by addition of 2N HCl. Thesolution was extracted with EtOAc (×2) and the combined organics weredried (MgSO₄) and concentrated in vacuo. The residue was passed througha silica plug, eluting first with DCM, then up to 5% MeOH in DCM.Fractions containing product were evaporated to a yellow solid (˜4.2 g).The residue (4.2 g) was dissolved in EtOH (20 ml) and warmed to 35° C.Water (30 ml) was added slowly over ˜40 mins. The mixture was thenstirred for another 30 minutes, then slowly cooled to room temperature.Additional water (30 ml) was added, and the reaction was then cooled to0° C. The mixture was filtered and the solids were washed with waterbefore being dried under vacuum at 35° C. overnight to afford(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoicacid (3.34 g, 73.3%) as a pale yellow solid. ¹H NMR (400 MHz, CDCl₃, 30°C.) 1.12 (3H, d), 1.19 (3H, d), 1.26 (3H, d), 2.43 (1H, dd), 2.63 (1H,dd), 2.87 (1H, dd), 3.07 (1H, dd), 3.65 (1H, q), 6.41 (1H, d), 7.02 (2H,d), 7.06-7.16 (2H, m), 7.19-7.25 (1H, m), 7.41 (1H, s), 7.48-7.57 (1H,m), 7.63 (1H, d), CO₂H not observed. m/z: ES+ [M+H]+ 444.

This compound could alternatively be named(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]acrylicacid.

Preparation of (4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol

Lithium borodeuteride (0.497 g, 17.25 mmol) was added portionwise to asolution of methyl 4-bromo-2,6-difluorobenzoate (2.89 g, 11.5 mmol) inTHF (46.0 ml). The reaction was heated to 50° C. for 2 hours. Aftercooling, (30 ml) 2N HCl was carefully added. The layers were separated,and the aqueous was extracted with EtOAc (2×50 ml). The combinedorganics were washed with brine, dried (MgSO₄) and concentrated toafford (4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol (2.250 g, 87%)as a white solid. ¹H NMR (400 MHz, CDCl₃, 30° C.) 1.96 (1H, s),7.07-7.13 (2H, m).

Preparation of 4-bromo-2,6-difluoro-1-deuterobenzaldehyde

Dess-Martin reagent (4.98 g, 11.73 mmol) was added to(4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol (2.20 g, 9.78 mmol) inDCM (39.1 ml) at room temperature. The reaction was stirred for 1 hour,then was quenched by addition of (50 ml) sat. NaHCO₃ containing 10%sodium thiosulfate. The layers were separated and the aqueous phase wasextracted with DCM (2×50 ml). The organics were dried (MgSO₄) andconcentrated, then the crude product was purified by flash silicachromatography, elution gradient 0 to 25% EtOAc in heptane. Purefractions were evaporated to dryness to afford4-bromo-2,6-difluoro-1-deuterobenzaldehyde (2.040 g, 94%) as a whitesolid. ¹H NMR (400 MHz, CDCl₃, 30° C.) 7.18-7.25 (2H, m). No mass ionobserved.

Preparation of methyl(E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate

4-Bromo-2,6-difluoro-1-deuterobenzaldehyde (3.33 g, 15.0 mmol),triethylamine (4.18 ml, 30.00 mmol), palladium (II) acetate (0.168 g,0.75 mmol) and tritolylphosphine (0.457 g, 1.50 mmol) were dissolved inDMF (36.6 ml), which was degassed. Methyl acrylate (2.026 ml, 22.50mmol) was then added and the reaction was heated to 80° C. for 4 hours.After cooling, the mixture was added to water (150 ml) and extractedwith EtOAc (2×150 ml). The combined organics were washed with 2N HCl(100 ml) then brine (100 ml), then dried (MgSO₄) and concentrated. Thecrude product was purified by flash silica chromatography, elutiongradient 0 to 40% EtOAc in heptane. Pure fractions were evaporated todryness to afford methyl(E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate (2.93 g,86%) as a yellow solid. 1H NMR (400 MHz, CDCl₃, 30° C.) 3.83 (3H, d),6.51 (1H, d), 7.12 (2H, m), 7.57 (1H, d). m/z (ES+), [M+H]+=228.

(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine

(2R)-1-(1H-indol-3-yl)propan-2-amine (3.81 kg, 21.21 moles) was added toa 100 L glass lined jacketed vessel under an atmosphere of nitrogen.1,4-dioxane (23 L) was added, and the agitator was switched on.Diisopropylethylamine (5.55 L; 31.82 moles) was added to the stirredsuspension followed by(2-fluoro-2-methyl-propyl)trifluoromethanesulfonate (5.55 kg, 23.77moles). 1,4-Dioxane (4 L) was added to the vessel, and the mixture washeated to 75° C. Heating was continued for 24 hours before cooling themixture to 25° C. Water (30.5 L) was added to the vessel, followed bytoluene (30.5 L). After 40 minutes the agitator was switched off and thelayers were allowed to separate. The aqueous layer was removed and water(30.5 L) was added to the organic solution. The mixture was agitated for15 minutes before allowing the layers to separate. The aqueous layer wasremoved from the vessel. The organic solution was concentrated by vacuumdistillation (jacket temperature 65° C., 110 mbar pressure) untilapproximately 27 L of distillate had been removed. The remainingsolution in the vessel was cooled to afford(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine asa solution in toluene (33% w/w) (15.4 Kg, 97%). 1H NMR (500 MHz, DMSO,27° C.) 0.98 (3H, d), 1.26 (3H, d), 1.30 (3H, d), 2.57-2.75 (3H, m),2.81 (1H, dd), 2.84-2.92 (1H, m), 6.97 (1H, t), 7.06 (1H, t), 7.11-7.22(1H, multiplet obscured by toluene signals), 7.34 (1H, d), 7.52 (1H, d),10.80 (1H, s).

Preparation of methyl(E)-3-[4-1[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate

(R)—N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine[33% w/w in toluene] (11.26 g, 14.97 mmol) and(E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate (3.40 g,14.97 mmol) were heated in toluene (55.6 ml)/acetic acid (4.28 ml, 74.83mmol) at 80° C. for 5 hr. After cooling, the volatiles were removedunder vacuum. The residue was taken-up in DCM (200 ml) and washed withsat. NaHCO₃ solution (200 ml). The aqueous phase was extracted with DCM(100 ml) then the combined organics were washed with brine, dried andconcentrated in vacuo. The crude material was loaded to an SCX-2 column,eluting with methanol to remove unreacted aldehyde. The column was theneluted with 7M NH₃-MeOH to liberate the product. The basic filtrate wasevaporated and the crude product was purified by flash silicachromatography, elution gradient 0 to 40% EtOAc in heptane. Purefractions were evaporated to dryness to afford methyl(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate(4.70 g, 68.6%) as a pale yellow solid.

¹H NMR (400 MHz, CDCl₃, 30° C.) 1.11 (3H, d), 1.19 (3H, d), 1.25 (3H,d), 2.42 (1H, dd), 2.62 (1H, dd), 2.87 (1H, dd), 3.07 (1H, dd), 3.65(1H, q), 3.81 (3H, s), 6.39 (1H, d), 6.99 (2H, d), 7.06-7.17 (2H, m),7.23 (1H, dd), 7.45 (1H, s), 7.49-7.6 (2H, m). m/z: ES+[M+H]+ 458.

Example 11 Preparation of(1R,3R)-1-{4-[(E)-2-carboxyethenyl]-2,6-difluorophenyl}-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-beta-carbolin-2-iummaleate

A solution of maleic acid (1.31 g, 11.29 mmol) in acetone (15 ml) wasstirred under nitrogen. A solution of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (Example 1) (5.00 g, 11.3 mmol) in acetone (25 ml) was added to themaleic acid solution to give a yellow solution. The reaction vessel wascovered in foil to protect from light and purged with a stream ofnitrogen gas overnight until the solvent evaporated. A solid wasobtained which was dried in vacuo for 2 hours to give the title compoundas a cream solid (6.23 g, 98%). ¹H NMR (500 MHz, DMSO, 27° C.) 0.95-1.34(9H, m), 2.24-2.45 (1H, m), 2.54-2.66 (1H, m), 2.8-2.99 (2H, m), 3.52(1H, s), 5.22 (1H, s), 6.26 (2H, s), 6.67 (1H, d), 6.89-7.07 (2H, m),7.19 (1H, d), 7.39-7.51 (3H, m), 7.55 (1H, d), 10.59 (1H, s).

An XRPD trace of this maleate salt is shown in FIG. 8 and a DSC traceshown in FIG. 9.

Example 12

Exemplary compositions of Example 1 were manufactured at the 75 g scaleusing a wet granulation process. The active ingredient, mannitol,microcrystalline cellulose and sodium starch glycolate were weighed inthe quantities tabulated below and transferred to a Diosna P1-6mixer-granulator and mixed (with chopping) at 600 rpm for 6 minutes. ForComposition A, mixing was continued while 30 mL water was added in twoaliquots at a rate of approximately 1 mL per second, pausing mixing inbetween aliquots, while for Composition B a solution prepared bystirring the required amounts of EDTA and ascorbic acid with 20 mL waterat 50° C. for 20 minutes (protected from light) was added using ananalogous process, the second aliquot in this case comprisingapproximately 10 mL of rinse liquor. Wet mixing was continued for atotal of 1.5 minutes. The wet granules were passed through a 1.5 mmscreen then dried under vacuum at 50-60° C. to a moisture content of <2%w/w. The resulting granules were milled using a 1 mm screen then mixedwith the lubricant for 5 minutes at 32 rpm using a Turbula blender.Tablets containing 10 mg of the active ingredient were formed bycompressing the granules to a nominal 100 mg compression weight using aRiva Mini-press equipped with 6 mm normal concave tooling.

Composition A Composition B % Amount % Amount Component Function w/w (g)w/w (g) Example 1 Active 10.0 7.50 10.0 7.50 Form B ingredient EDTAChelating Not present 0.1 0.075 agent Ascorbic Anti- Not present 0.50.375 acid oxidant Mannitol Diluent 68.0 51.00 67.4 50.55Microcrystalline Diluent 15.0 11.25 15.0 11.25 cellulose Sodium starchDisintegrant 5.0 3.75 5.0 3.75 glycolate Stearic acid Lubricant 2.0 1.502.0 1.50 Total 100 75.00 100 75.00

The stability of Compositions A and B with regard to degradant formation(wherein “degradant” means(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate)was evaluated for tablets packed in induction sealed 75 cc HDPE bottlesor exposed to the atmosphere in petri dishes, stored in the dark, undercontrolled temperature and humidity as tabulated below. It is apparentthat Composition B, which contains both a chelating agent andanti-oxidant, is more stable to chemical degradation than Composition Awhich is a standard tablet formulation.

Stability of exemplary compositions with regard to Degradant formation(%) 4 Week Time Point (storage condition) Initial (25° C./ (25° C./ (40°C./ Time (5° C., 60% RH, 60% RH, 75% RH, Formulation Point packed)packed) exposed) exposed) Composition A 0.05 0.06 0.08 0.27 0.28Composition B ND <0.05 <0.05 0.05 0.11 ND Not detected (<0.02% w/w)

Example 13(E)-3-[3,5-difluoro-4-[(3R)-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate

(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (200 mg, 0.45 mmol) was added to a solution of cerium ammoniumnitrate (248 mg, 0.45 mmol) in acetonitrile (6 ml)/water (1.500 ml) atroom temperature. The reaction was stirred for 2 hr and further ceriumammonium nitrate (248 mg, 0.45 mmol) was added. The solution was stirredat 25° C. for a further 15 minutes. The reaction mixture was acidifiedwith 2M HCl (3 ml) and extracted with DCM (2×10 ml). The organics werethen concentrated in vacuo and the crude product was purified bypreparative HPLC (Waters SunFire column, 5. silica, 50 mm diameter, 100mm length), using decreasingly polar mixtures of water (containing 0.1%formic acid) and MeCN as eluents. Fractions containing the desiredcompound were evaporated to dryness to afford(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate(35.0 mg, 17.58%) as an orange glass. 1H NMR (500 MHz, DMSO, 30° C.)1.27 (3H, d), 1.43-1.55 (6H, m), 3.51 (1H, d), 3.72 (1H, dd), 3.96 (1H,dd), 4.18-4.32 (1H, m), 4.67 (1H, s), 6.59 (1H, s), 7.09-7.3 (2H, m),7.42 (1H, t), 7.52-7.73 (3H, m), 7.78 (1H, d), NH not observed. HRMS(ESI): [M+H]⁺, found 441.17831, C₂₃H₂₄F₂N₂O₂ requires 441.17844.

Preparation of(E)-3-[3,5-difluoro-4-[2-(2-fluoro-2-methyl-propyl)-3-methyl-9H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate

(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (0.500 g, 1.13 mmol) was dissolved in DMSO (10 mL) and heated to120° C. in air and light for 16 h. The reaction was then heated to 180°C. for 2.5 h. The reaction mixture was cooled and purified bypreparative HPLC (Waters XBridge Prep C18 OBD column, 5 μ silica, 50 mmdiameter, 100 mm length), using decreasingly polar mixtures of water(containing 1% NH₃) and MeCN as eluents. Fractions containing thedesired compound were evaporated to dryness to afford(E)-3-[3,5-difluoro-4-[2-(2-fluoro-2-methyl-propyl)-3-methyl-9H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate(0.047 g, 9.40%) as an orange solid. 1H NMR (400 MHz, DMSO, 27° C.) 1.27(3H, s), 1.33 (3H, d), 3.09 (3H, s), 4.59-4.8 (1H, m), 5.14-5.37 (1H,m), 6.54 (1H, d), 7.19 (1H, d), 7.39 (1H, t), 7.63 (2H, d), 7.74 (1H,t), 7.91 (1H, d), 8.45 (1H, d), 8.88 (1H, s), 10.79 (1H, s). m/z: ES+[M+H]+ 439.

Example 14A Preparation of(E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-3-hydroxy-2-methyl-propyl)-3-methyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoicacid (Isomer 1)

2M Sodium hydroxide (1.27 mL, 2.54 mmol) was added to a solution of(E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate-Isomer1 (120 mg, 0.25 mmol) in THF (0.635 mL)/methanol (0.635 mL). Thereaction was stirred at room temperature for 1 h, then diluted withEtOAc and water. The aqueous was adjusted to pH 6 by addition of 2M HCl,and the layers were separated. The aqueous layer was extracted withEtOAc, then the combined organics were dried (MgSO₄) and concentrated invacuo. The crude product was purified by flash silica chromatography,elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated todryness to afford(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid-Isomer 1 (85 mg, 72.9%) as a beige solid. 1H NMR (400 MHz, DMSO,27° C.) 0.97 (3H, d), 1.05 (3H, d), 2.32-2.4 (1H, m), 2.44-2.54 (1H, m),2.73-2.93 (3H, m), 3-3.14 (2H, m), 3.32-3.49 (2H, m), 4.73 (1H, s), 5.14(1H, s), 6.58 (1H, d), 6.82-6.96 (2H, m), 7.10 (1H, d), 7.33 (1H, d),7.36 (1H, d), 7.45 (1H, d), 10.49 (1H, s). m/z (ES+), [M+H]+=459.

Example 14B Preparation of(E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-3-hydroxy-2-methyl-propyl)-3-methyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoicacid (Isomer 2)

2M Sodium hydroxide (1.27 mL, 2.54 mmol) was added to a solution of(E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate-Isomer2 (110 mg, 0.23 mmol) in THF (0.529 mL)/methanol (0.529 mL). Thereaction was stirred at room temperature for 1 h, then diluted withEtOAc and water. The aqueous was adjusted to pH 6 by addition of 2M HCl,and the layers were separated. The aqueous layer was extracted withEtOAc, then the combined organics were dried (MgSO₄) and concentrated invacuo. The crude product was purified by flash silica chromatography,elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated todryness to afford(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid-Isomer 2 (81 mg, 76%) as a beige solid. 1H NMR (400 MHz, DMSO, 27°C.) 1.02 (2H, s), 1.05 (3H, d), 1.23 (1H, s), 1.90 (3H, s), 2.28-2.46(1H, m), 2.53-2.7 (1H, m), 2.86-3.03 (2H, m), 3.56 (1H, d), 4.83 (1H,s), 5.17 (1H, s), 6.66 (1H, d), 6.86-7.08 (2H, m), 7.17 (1H, d), 7.40(1H, d), 7.44 (2H, d), 7.53 (1H, d), 10.54 (1H, s). m/z (ES+),[M+H]+=459.

The (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(Isomer 1 and 2) used as starting materials were prepared as follows:—

2-Fluoro-2-methylpropane-1,3-diol

LiAlH₄ (0.741 g, 19.25 mmol) was added portionwise to a cooled solutionof diethyl 2-fluoro-2-methylmalonate (1.345 g, 7.00 mmol) in THF (35.0ml). The reaction was allowed to warm to room temperature over 1 h.After cooling to 0° C., the reaction was quenched by addition of water(0.75 mL), 15% NaOH (0.75 mL), then water (1.5 mL). The suspension wasstirred for 30 min, then filtered and the solids were washed with THF.The filtrate was evaporated to afford 2-fluoro-2-methylpropane-1,3-diol(0.745 g, 98%) as a colourless oil. 1H NMR (400 MHz, CDCl₃, 27° C.) 1.34(3H, d), 2.12-2.27 (2H, m), 3.75 (4H, d).

3-(((R)-1-(1H-Indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol

Trifluoromethanesulfonic anhydride (1.151 ml, 6.80 mmol) was added to asolution of 2-fluoro-2-methylpropane-1,3-diol (0.70 g, 6.47 mmol) in DCM(17.85 ml) at 0° C., followed by 2,6-lutidine (0.908 ml, 7.77 mmol). Thereaction was allowed to warm to room temperature over 30 min, then waswashed with 2M HCl. The organic phase was passed through a phaseseparator cartridge and concentrated in vacuo. The residue was dissolvedin dioxane (12 mL), then (R)-1-(1H-indol-3-yl)propan-2-amine (1.128 g,6.47 mmol) and DIPEA (1.678 ml, 9.71 mmol) were added and the reactionwas heated to 90° C. for 2 h. After cooling, the reaction was dilutedwith DCM and washed with water. The aqueous was extracted with DCM, thenthe organics were concentrated in vacuo. The crude product was purifiedby flash silica chromatography, elution gradient 0 to 10% MeOH in DCM.Pure fractions were evaporated to dryness to afford3-(((R)-1-(1H-indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol(0.815 g, 47.6%) as a brown gum. m/z (ES+), [M+H]+=265.

(E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(Isomer 1 and 2)

(E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (565 mg, 2.50 mmol)was added to a suspension of3-(((R)-1-(1H-indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol(661 mg, 2.50 mmol) in toluene (11.3 ml)/acetic acid (1.25 ml). Thereaction was heated to 90° C. for 5 h. After cooling, the volatiles wereremoved under vacuum, then the residue was passed through an SCX-2column, eluting with methanol to remove unreacted aldehyde. The columnwas then eluted with NH₃/MeOH to liberate the products. The basicfiltrate was evaporated then the crude product was purified by flashsilica chromatography, elution gradient 0 to 50% EtOAc in heptane. Purefractions were evaporated to dryness to afford (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(Isomer 1-122 mg, 10.3%) as a yellow solid and (E)-methyl3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate(Isomer 2-129 mg, 11% as a yellow/orange solid. Isomer 1-1H NMR (400MHz, CDCl₃, 27° C.) 1.10 (3H, d), 1.14 (3H, d), 2.54 (1H, dd), 2.62-2.73(1H, m), 3.06-3.3 (2H, m), 3.40 (1H, dd), 3.56 (1H, t), 3.80 (3H, s),3.87-4.08 (1H, m), 4.27 (1H, s), 5.16 (1H, s), 6.37 (1H, d), 7.01 (2H,d), 7.07-7.15 (2H, m), 7.14-7.24 (1H, m), 7.42-7.56 (2H, m), 7.71 (1H,s). m/z (ES+), [M+H]+=473. Isomer 2-1H NMR (400 MHz, CDCl3, 27° C.) 1.15(3H, d), 1.20 (3H, d), 2.65 (1H, dd), 2.79 (1H, t), 2.93-3.09 (2H, m),3.57 (1H, dt), 3.70 (1H, dd), 3.78 (3H, s), 4.2-4.67 (1H, m), 5.42 (1H,s), 6.32 (1H, d), 6.94 (2H, d), 7.06-7.15 (2H, m), 7.19-7.27 (2H, m),7.42 (1H, s), 7.51 (1H, dd), 8.02 (1H, s). m/z (ES+), [M+H]+=473.

Reference Example 11-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

Pyridine 4-methylbenzenesulfonate (11.62 g, 46.24 mmol) was added to asuspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(128 g, 231.19 mmol) in methanol (1 L) under nitrogen. The mixture wasstirred at 60° C. for 1.5 hours. The mixture was soluble after 5minutes. The mixture was held at 50° C. overnight during which time aprecipitate formed. The solid material was isolated by filtration andwashed with water and acetonitrile. This material still contained minorimpurities from the previous stage and required further purification.The material was dissolved in dichloromethane and purified by flashchromatography on silica gel (0% methanol/DCM to 10% methanol/DCM). Thedesired product,1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(Reference Example 1) (92 g, 85%), was thus isolated as a cream solid(Form A): ¹H NMR Spectrum: (DMSO-d₆) 1.4-1.51 (12H, m), 1.51-1.78 (2H,m), 1.89-2.05 (2H, m), 2.72-2.86 (1H, m), 2.91-3.05 (1H, m), 3.12-3.24(1H, m), 3.64 (2H, q), 3.83-4.01 (1H, m), 4.29-4.41 (1H, m), 4.47 (1H,t), 4.58 (2H, q), 8.26 (2H, s), 8.85 (1H, s); Mass Spectrum [M+H]⁺=470.

1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-onewas prepared as follows:

1,8-Diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) was added to asuspension of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(150 g, 319.46 mmol) in 2-methyl THF (1.2 L). Iodoethane (46 mL, 575.03mmol) was added and the mixture was stirred for 16 hours at 35° C.Further iodoethane (46 mL, 575.03 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) were added andstirring was continued for 24 hours at 35° C. The mixture was pouredinto water and the insoluble material was isolated by filtration, washedwith water and MTBE and dried in vacuo to afford tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(116 g, 73.0%) as a yellow solid. The filtrate was extracted with DCMand the organic solution was dried with magnesium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica using gradient elution (30% MTBE/heptaneto 100% MTBE). A second crop of the desired product (12 g, 24.12 mmol,7.55%), was thus isolated as a yellow solid which was later combinedwith the first crop: ¹H NMR Spectrum: (DMSO-d₆) 1.41 (9H, s), 1.44 (9H,s), 1.48 (3H, t), 1.52-1.69 (2H, m), 1.87-2.04 (2H, m), 2.79-3.03 (3H,m), 3.86-4.03 (2H, m), 4.59 (2H, q), 7.89 (2H, s), 8.85 (1H, s); MassSpectrum [M+H]⁺=498.

A suspension of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(3009.5 g, 6.05 mol) in DCM (9 L) was cooled to 5-10° C. under N₂. TFA(9 L) was added portionwise to the suspension whilst maintaining thetemperature <30° C. The reaction mixture was stirred at room temperaturefor 1 h. The mixture was concentrated, the resulting residue wasdissolved in water (30 L) and added slowly to a 35% aqueous ammoniasolution (12 L) at 0-5° C. The suspension was stirred for 30 min thenthe product was filtered off and washed with water (2×6 L). The productwas dried at 50° C. in vacuo for 2 days. to afford3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine((2496 g): ¹H NMR Spectrum: (DMSO-d₆) 1.4-1.52 (12H, m), 1.57-1.73 (2H,m), 1.83-1.93 (2H, m), 2.57-2.7 (2H, m), 2.71-2.84 (1H, m), 2.96-3.09(2H, m), 4.58 (2H, q), 8.06 (2H, s), 8.84 (1H, s); Mass Spectrum[M+H]⁺=398.

To a solution of 3-(tetrahydro-2H-pyran-2-yloxy) propanoic acid (HATU,48.80 g 0.2774 mol) and N-ethyl-N-isopropylpropan-2-amine (86.96 mL,0.4993 mol) in THF (552 mL) was addedO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (115.73 g, 0.3051 mol) portionwise at RT undernitrogen. The resulting mixture was stirred for 20 min then3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(122.5 g (110.25 g active), 0.2774 mol) was added portionwise over 1 h.After 3.5 h, the mixture was concentrated and the residue was slurriedin MeCN (275 mL) for 15 min at room temperature. The product wasfiltered off, washed with MeCN (3×110 mL) and dried overnight at 50° C.in vacuo. This gave1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(131.9 g, 96%). ¹H NMR Spectrum: (DMSO-d₆) 1.29-1.48 (16H, m), 1.48-1.75(4H, m), 1.83-1.99 (2H, m), 2.48-2.68 (2H, m), 2.68-2.79 (1H, m),2.87-2.99 (1H, m), 3.07-3.19 (1H, m), 3.32-3.42 (1H, m), 3.47-3.6 (1H,m), 3.64-3.75 (1H, m), 3.75-3.84 (1H, m), 3.84-3.95 (1H, m), 4.24-4.39(1H, m), 4.47-4.6 (3H, m), 7.84 (2H, s), 8.79 (1H, s): Mass Spectrum[M+Na]⁺=577.

Alternative Preparation of Reference Example 1

To a suspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(131.9 g, 0.2382 mol) in methanol (1045 mL) was added pyridiniump-toluenesulfonate (11.97 g, 47.7 mmol) under N₂. The reaction mixturewas stirred at 60° C. for 5.5 h then at 50° C. overnight. The reactionmixture was cooled to 0° C. and the solid was filtered off. The productwas slurried in water (250 mL) for 20 min at room temperature, filteredoff, washed with water (3×40 mL) and dried at 50° C. in vacuo. This gave1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(21.4 g) as Form A (see below).

The methanol liquors were concentrated and the resulting solid wasslurried in water (0.6 L) for 20 min at room temperature. The solid wasisolated by filtration and washed with water (3×100 mL). The filter cakewas slurried for a second time in water (0.5 L) for a further 20minutes. The product was isolated by filtration, washed with water (100mL) and dried at 50° C. in vacuo. This gave 81.9 g1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(81.9 g) as Form A.

Both crops were combined (103.3 g), seeded with Form B (16.68 g) andslurried in MeCN (826 mL) at room temperature overnight. This gave 117.4g of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-oneas a pale yellow solid (117.4 g), Form B (see below). This material wasfurther purified by slurrying in heptane (7.5 rel vols) for 1 hour. Themixture was filtered, pulled dry on the filter, and dried at 50° C. in avacuum oven overnight to afford1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(102.5 g) as Form B.

Form B may also be made by slurrying Form A in MeCN without seeding.

Form A or B may also be converted to Form C as follows:

A suspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(eg Form B made by the processes outlined above) in IPA (12 vol) washeated at reflux until the solid dissolved. The solution was hotfiltered then cooled to room temperature. This gave1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-oneas a pale yellow solid (99.3 g, 97%) as Form C.

Form C may also be converted to Form B as follows:

In a 10 L flange flask, Form C (377.8 g portion 1) in MIBK (7900 mL) washeated to 110-115° C. to give a solution. The solution was allowed tocool to 97-103° C. and immediately polish filtered into a 50 L vesselcontaining a seed of Form B (0.8 g) in acetonitrile (8220 mL) stirringat −15° C. During the addition the temperature in the 50 L vessel wasmaintained between −15 and 25° C. by means of jacket cooling. Threefurther portions of the compound dissolved in MIBK were added by asimilar method. To the resulting slurry was added a seed of form B (0.8g) and the mixture was then stirred at 10-20° C. overnight. In-processanalysis confirmed the desired form (Form B) with no Form C or amorphousvisible. The mixture was filtered and washed with acetonitrile (3340mL). The solid was oven dried for 2 days (solid was broken up during thedrying to a powder and a mixture of small lumps ˜1 mm to ˜3-4 mm size)until constant weight was obtained. Yield=1532.8 g (93.5%)

3-(Tetrahydro-2H-pyran-2-yloxy)propanoic acid was prepared as follows:

To a stirred solution of methanol (2.4 L) and concentrated sulfuric acid(44.4 mL, 832.61 mmol) at 0° C. under nitrogen was added, dropwise,beta-propiolactone (175 mL, 2.78 mol). This solution was allowed to stirat room temperature for 2 days. The reaction mixture was cooled to 10°C. before adding, portionwise, sodium bicarbonate (145 g, 1.72 mol), theresulting suspension was left to stir at room temperature for 75minutes. This solution was filtered, the filter-cake was washed withmethanol (800 mL). The filtrate was evaporated to an oil which wasredissolved in dichloromethane (1.2 L) and stirred for 60 minutes beforerefiltering. This solution was filtered before evaporating to givemethyl 3-hydroxypropanoate (219 g, 76%) as an oil. ¹H NMR Spectrum:(CDCl₃) 2.50 (2H, t), 3.63 (3H, s), 3.78 (2H, t).

Pyridinium p-toluenesulfonate (7.65 g, 30.45 mmol) was added to a clearsolution of methyl 3-hydroxypropanoate (63.4 g, 609.00 mmol) and3,4-dihydro-2H-pyran (78 mL, 852.60 mmol) in dichloromethane (650 mL) atroom temperature under nitrogen to give a cloudy solution. This wasallowed to stir at room temperature overnight. The reaction mixture waswashed with water (250 mL) and brine (250 mL) before drying (MgSO₄) andevaporating to an oil. This crude product was purified by flash silicachromatography, elution gradient 15 to 30% EtOAc in heptane. Purefractions were evaporated to dryness to afford methyl3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.7 g, 59.0%) as acolourless oil: ¹H NMR Spectrum: (CDCl₃) 1.47 (4H, dddd), 1.55-1.84 (2H,m), 2.55 (2H, t), 3.33-3.53 (1H, m), 3.53-3.7 (4H, m), 3.78 (1H, ddd),3.93 (1H, dt), 4.42-4.72 (1H, m); Mass Spectrum [MH]⁺=189.

Sodium hydroxide (2M, 349 mL, 697.58 mmol) was added to a solution ofmethyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.68 g, 359.58 mmol)in THF (680 mL) at room temperature. The reaction mixture was stirred atroom temperature for 3 hours. The THF was removed in vacuo, the aqueouslayer was then washed with ethyl acetate (260 mL), before cooling to 0°C. and careful acidification to pH 5 by the addition of hydrochloricacid (2M). The product was extracted with ethyl acetate (3×250 mL)before drying (MgSO₄) and evaporation to give3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (57.0 g, 91%) as a clearoil. This material was dissolved in ethyl acetate (750 mL) then washedwith water (3×250 mL) and brine (250 mL) to remove remaining aceticacid. The organic solution was dried (MgSO₄) and evaporated to give3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (45.67 g, 72.9%) as acolourless oil: ¹H NMR Spectrum: ¹H NMR (CDCl₃) 1.43-1.67 (4H, m),1.65-1.95 (2H, m), 2.68 (2H, t), 3.48-3.58 (1H, m), 3.73 (1H, dt), 3.88(1H, ddd), 4.02 (1H, dt), 4.59-4.7 (1H, m); Mass Spectrum [M−H]⁻=173.

The tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylatewas prepared as follows:

Hydrazine hydrate (23.59 mL, 480.75 mmol) was added dropwise to astirred mixture of methyl 3-amino-6-bromopyrazine-2-carboxylate (100 g,418.04 mmol) in EtOH (2 L). The mixture was heated at 50° C. undernitrogen. The resulting thick suspension was stirred at 50° C. for 16hours. Further hydrazine (2.5 mL) was added in one portion and thesuspension was stirred at 50° C. for a further 24 hours. Ethanol (500mL) was charged to the thick reaction mixture and the mixture wasallowed to cool to room temperature. The resulting suspension wasfiltered and the solid washed with ethanol (1 L) and dried in vacuo togive 3-amino-6-bromopyrazine-2-carbohydrazide (98 g, quantitative) as acream solid: ¹H NMR Spectrum; (DMSO-d₆) 4.52 (2H, s), 7.59 (2H, s), 8.30(1H, s), 9.74 (1H, s); Mass Spectrum [M+H]⁺=232.

Pivalic anhydride (165 mL, 815.38 mmol) was added to a stirred mixtureof 3-amino-6-bromopyrazine-2-carbohydrazide (172 g, 741.26 mmol) inacetonitrile (1.8 L) and the mixture was heated at 80° C. for 1 hour.The reaction was left to stir for 16 hours. The required yellow solidmaterial was isolated by filtration. The filtrate was partitionedbetween EtOAc (2 L) and aqueous sodium bicarbonate (2 L). The organiclayer was washed with saturated brine and dried over MgSO₄. The solutionwas filtered and concentrated to give an orange sticky solid which wastriturated with MTBE (250 mL). The insoluble yellow solid was isolatedby filtration and this material was shown to be identical to the firstsolid. The combined solids were dried in the vacuum oven at 50° C. for 3days to afford 3-amino-6-bromo-N′-pivaloylpyrazine-2-carbohydrazide (224g, 96%) as a yellow solid: ¹H NMR Spectrum: (DMSO-d₆) 1.17 (9H, s), 7.62(2H, s), 8.37 (1H, s), 9.42-9.56 (1H, m), 10.09-10.23 (1H, m); MassSpectrum [M+H]⁺=318.

To 3-amino-6-bromo-N′-pivaloylpyrazine-2-carbohydrazide (2301 g, 7.28mol) in MeCN (10.8 L) was added DIPEA (3.044 L, 17.48 mol) andp-toluenesulfonyl chloride (1665 g, 8.73 mol) portion-wise (˜280 g×6) at50° C. over a period of 30 mins. The reaction temperature was maintainedbetween 65-70° C. by controlling the rate of addition. After theaddition was complete, the reaction mixture was stirred at 70° C. for 1h. The mixture was cooled to room temperature and quenched with 5%NaHCO₃ (aqueous, 24.2 L). The resulting suspension was stirred for 30min then filtered. The product was washed with water (14.8 L), pulleddry and dried at 50° C. for 16 h. The product was dissolved in DCM (12L) and the phases separated. The organic phase was loaded onto a silicapad (6 kg) and the product was eluted with 20% EtOAc/DCM (8×10 L).Concentration of the product containing fractions gave 1987 g (92%yield) with a purity of 99.8% by HPLC of5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (36 g,17%): ¹H NMR Spectrum: (DMSO-d₆) 1.43 (9H, s), 7.70 (2H, s), 8.39 (1H,s); Mass Spectrum [M+H]⁺=298.

A stream of nitrogen gas was passed through a solution of5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (89.35 g,239.75 mmol) in DMA (1.2 L) for 20 minutes.Dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine (11.43 g,23.98 mmol), tris(dibenzylideneacetone)dipalladium(0) (5.49 g, 5.99mmol), zinc (1.568 g, 23.98 mmol) and dicyanozinc (16.89 g, 143.85 mmol)were added sequentially to the stirred mixture. The mixture was heatedto 100° C. and stirred for 1 hour. The mixture was cooled andpartitioned between DCM (3 L) and water (1 L). The black mixture wasfiltered through celite and the organic layer was separated. Thesolution was washed with water then brine. The solution was dried withmagnesium sulfate and concentrated under reduced pressure. The residuewas triturated with MTBE and isolated by filtration, washing with MTBE.The filter cake was dried in vacuo to afford5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile(55.7 g, 95%) as a pale orange solid: ¹H NMR Spectrum: (DMSO-d₆) 1.46(9H, s), 6.02 (1H, s), 8.38 (2H, s); Mass Spectrum [M−H]⁻⁼242.

Hydrazine hydrate (82 mL, 1.69 mol) was added to5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile (55g, 225.18 mmol) in IPA (200 mL) and the mixture was heated at 50° C.under nitrogen for 16 hours. The mixture was cooled in an ice bath. Theresulting precipitate was collected by filtration, washed with IPA anddiethyl ether and dried to a constant weight to afford(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide(49.2 g, 79%) as a yellow solid: ¹H NMR Spectrum: (DMSO-d₆) 1.45 (9H,s), 5.26 (2H, s), 5.58 (2H, s), 7.56 (2H, s), 8.75 (1H, s); MassSpectrum [M+H]⁺=277.

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (74.3 g, 195.44 mmol) was added to a solution ofN-Boc-isonipecotic acid (41.1 g, 179.15 mmol) and 4-methylmorpholine(35.9 mL, 325.74 mmol) in DMA (800 mL). The mixture was stirred for 10minutes then(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide(45 g, 162.87 mmol) was added to the solution in one portion (exothermobserved from 22° C. to 27° C.). After a few minutes the productcrystallised from the reaction mixture. The reaction mixture was removedfrom the vessel and filtered through a sinter. Additional DMA was addedto wash product from the sides of the vessel (150 mL) and this waspoured onto the filter cake. Isopropanol (600 mL) was added to thevessel and the remainder of the product in the vessel was suspended inthis solvent using vigorous agitation. The isopropanol suspension wasused to wash the filter cake once the DMA had been removed by suction.The filter cake was sucked dry then washed with MTBE and sucked dry onceagain. The filter cake was dried in vacuo to afford (Z)-tert-butyl4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)piperidine-1-carboxylate(76 g, 95%) as a yellow solid: ¹H NMR Spectrum: (DMSO-d₆) 1.40 (9H, s),1.46 (9H, s), 1.63-1.9 (2H, m), 2.33-2.6 (2H, m, obscured by DMSOsignal), 2.63-3.03 (2H, m), 3.18-3.48 (4H, m, obscured by water signal),3.88-4.11 (2H, m), 6.43 (2H, s), 7.76 (2H, br), 8.84 (0.5H, s), 8.87(0.5H, s), 9.85 (1H, s); Mass Spectrum [M+H]⁺=488

In an alternative preparation, the N-Boc-isonipecotic acid may be madein situ as follows: Isonipecotic acid (858 g, 3.74 mol) was dissolved inDMA (25.3 L) and 4-methylmorpholine (393 mL, 3.74 mol) added. Stirredfor 5 mins and isobutyl chloroformate (489 mL, 3.74 mol) added. Thereaction mixture was stirred at 25° C. for 2 h and cooled to 15° C.before(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide(940 g, 3.4 mol) was added portionwise over 10 mins. The reactionmixture was stirred for 1-2 h at 15° C. Water (20.5 L) was addedportionwise over 1 h and stirred for a further 1 h before beingfiltered. The filtercake was then washed with water (4×4 L) and pulleddry on the filter before being dried in a vacuum oven at 50° C. untildry to give the desired product.

Acetic acid (200 mL) was added to dioxane (500 mL) in a 3 L fixed doublejacketed vessel and the solution was heated to 70° C. under nitrogen.(Z)-tert-butyl4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)-piperidine-1-carboxylate(74.5 g, 152.80 mmol) was added portionwise to the warm mixture. After10 minutes the temperature was increased to 100° C. (slight reflux). Thereaction mixture was stirred at 100° C. for 1.5 hours (suspension) thenheld at 80° C. overnight (solution formed after overnight hold). Theresulting solution was concentrated under reduced pressure, then dilutedwith toluene, evaporated to dryness, taken up with toluene andconcentrated again. The residual oil was mixed with some ethyl acetateand concentrated to dryness. A solid crystallised from solution whichwas triturated with MTBE (200 mL) and isolated by filtration. The filtercake was washed with water and MTBE to afford tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(50 g, 70%) as a grey solid.

The filtrate was concentrated under reduced pressure to give a yellowsolid. This material was triturated with MTBE and filtered. The filtercake was washed with ethyl acetate and then MTBE to give a second cropas a pale yellow solid (4.93 g, 7%). This material was identical to thefirst crop: ¹H NMR Spectrum: (DMSO-d₆) 1.17 (9H, s), 1.22 (9H, s),1.29-1.47 (2H, m), 1.67-1.78 (2H, m), 2.57-2.87 (3H, m), 3.57-3.92 (2H,m), 7.56 (2H, br), 8.56 (1H, s), 13.47 (2H, br s); Mass Spectrum[M+H]+=470.

The invention claimed is:
 1. A method for the treatment of a cancerselected from breast cancer and a gynecological cancer in a warm bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of the compound(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyI)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid, or a pharmaceutically acceptable salt thereof.
 2. The methodaccording to claim 1, wherein the compound is(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyI)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.
 3. The method according to claim1, wherein the compound is the maleic acid salt of(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyI)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid.
 4. The method according to claim 1, wherein the cancer is breastcancer.
 5. The method according to claim 1, wherein the breast cancer isER+ve breast cancer.
 6. A method for the treatment of a cancer selectedfrom breast cancer and a gynecological cancer in a warm-blooded animal,such as man, in need of such treatment which comprises administering tosaid animal an effective amount of the compound(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid or a pharmaceutically acceptable salt thereof, in combination withanother anti-tumour agent.
 7. The method according to claim 6, whereinthe compound is(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.
 8. The method according to claim6, wherein the compound is the maleic acid salt of(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid.
 9. The method according to claim 6, wherein the anotheranti-tumour agent is palbociclib.
 10. The method according to claim 6,wherein the cancer is breast cancer.
 11. The method according to claim6, wherein the breast cancer is ER+ve breast cancer.
 12. The methodaccording to claim 6, wherein the another anti-tumour agent is selectedfrom an mTOR inhibitor or a cyclin dependent kinase inhibitor.
 13. Themethod according to claim 6, wherein the another anti-tumour agent isAZD2014.